Calcium Oxalate Crystal Deposition Research Articles

Rapidly progressive renal failure associated with successful pharmacotherapy for obesity

A 55-year-old woman presented with recurrent episodes of hypoglycaemia over 4 weeks. She had had type 1 diabetes mellitus (DM) for 35 years, managed by an insulin basal bolus regime. Her comorbidities included retinopathy, chronic renal failure, (presumed to be on the basis of diabetic nephropathy), hypertension, obesity, gout and an ischaemic stroke 15 years earlier. Over 5 years of follow-up, her renal function showed a stable decline of 3ml/min/year, with a creatinine of 141mmol/l and estimated glomerular filtration rate (eGFR) of 66ml/min/1.73m 5 months before presentation (Figure 1). She self-regulated an insulin basal bolus regime and hypoglycaemic episodes had been rare. Her other medication included a statin, six anti-hypertensive agents, warfarin and orlistat. Orlistat had been commenced 5 months previously with a subsequent 12 kg weight reduction. There had been no other new medication. Clinical examination was unremarkable other than a body mass index (BMI) of 35 kg/m and a blood pressure of 176/86mmHg. Initial investigations revealed advanced renal failure with a serum creatinine of 626 mmol/l, eGFR 12ml/ min/1.73m, metabolic acidosis and hyperkalaemia (potassium 6.8mmol/l). Urinalysis revealed only proteinuria. An ultrasound scan showed that both kidneys were non-obstructed and anatomically normal. The bladder was empty. Her inflammatory markers were normal. Immunological investigations were negative. Despite preservation of urine output, there was no improvement in renal function, and persistent hyperkalaemia necessitated haemodialysis. After reversal of anticoagulation, renal biopsy showed diabetic nephropathy with moderate interstitial scarring and widespread glomerulosclerosis. Additionally there was extensive, superimposed, intra-tubular deposition of calcium oxalate crystals in the kidney, with mild to moderate tubulo-interstitial inflammation (Figure 2). There was no personal or family history of renal stone disease or traditional risk factors for calcium oxalate crystallization. A 2-week course of oral steroid therapy was initiated to reduce the inflammatory component of the renal disease, without objective improvement. There was no recovery of renal function and 24 months later she remains dialysis-dependent.

Acquired cystic disease-associated renal tumors: an immunohistochemical and fluorescence in situ hybridization study

End-stage renal disease is associated with an increased incidence of renal cell neoplasms. Among these, recent studies have identified tumors with unusual histological patterns that do not fit into the categories recognized in the current classification system. These tumors often occur in kidneys with acquired cystic disease and are composed mainly of large eosinophilic cells arranged in solid, cribriform, acinar, or papillary patterns. They also contain deposits of calcium oxalate crystals. We investigated three eosinophilic epithelial tumors arising in kidneys with acquired cystic disease from three patients. Each of the tumors was composed of large eosinophilic cells arranged in solid, acinar, or tubulocystic architecture. Deposits of calcium oxalate crystals were present in each tumor. Hale's colloidal stain showed a positive cytoplasmic reaction in one of the neoplasms. Immunohistochemistry displayed positive results for CD10 (3/3), AE1/AE3 (3/3), alpha-methylacyl-CoA racemase (2/3), CAM5.2 (2/3), and vimentin (1/3). Reactions for epithelial membrane antigen, cytokeratin 7, and high molecular weight cytokeratin (34βE12) were negative. Fluorescence in situ hybridization analysis showed no losses or gains of chromosomes 1, 2, 6, 10, or 17 in one tumor. There were gains of chromosomes 1, 2, and 6 in two tumors. One of these tumors also showed gains of chromosome 10. Eosinophilic renal cell tumors associated with acquired cystic disease have immunophenotypes and genetic profiles distinct from the renal cell neoplasms recognized in the current classification of renal cell neoplasia, and should be considered as a distinct clinicopathologic entity in the spectrum of renal cell neoplasia.

LEAF ANATOMY OF CONOPHYTUM N. E. BR. (AIZOACEAE)

Leaf anatomy of Conophytum–a genus of 100 species of miniature xerophytic plants from the succulent Karoo in southern Africa–was examined using thin sections of plastic-embedded tissue and bright-field and polarized-light microscopy. Eighty-four species, from all recognized sections of the genus, were sectioned using material of known wild origin, with many species represented by multiple infraspecific taxa or populations. The broad scope of this investigation permits, for the first time, a meaningful study of the comparative anatomy of this large, morphologically diverse genus. The distribution of anatomical characters is described including: the pattern of deposition of calcium oxalate crystals in the epidermis, the presence and number of layers of hypodermis, and the presence and shape of tanniniferous idioblasts in the mesophyll. Typically xeromorphic characters like a hypodermis and epidermal crystal layers tend to be reduced or absent in species from sheltered niches, including soil-embedded, subterranean species with windowed leaves. Embedded species also frequently lack tannin cells, perhaps because their growth form reduces the need for chemical defenses against herbivores.

Expression of osteopontin in rat kidneys: induction during ethylene glycol induced calcium oxalate nephrolithiasis.

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. 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. 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. 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.

Early renal failure after domino hepatic transplantation using the liver from a compound heterozygous patient with primary hyperoxaluria

To cover the shortage of cadaveric organs, new approaches to expand the donor pool are needed. Here we report on a case of domino liver transplantation (DLT) using an organ harvested from a compound heterozygous patient with primary hyperoxaluria (PHO), who underwent combined liver and kidney transplantation. The DLT recipient developed early renal failure with oxaluria. The time to the progression to oxalosis with renal failure in such situations is unknown, but, based on animal data, we hypothesize that calcineurin inhibitors may play a detrimental role. A cadaveric liver and kidney transplantation was performed in a 52-year-old male with PHO. His liver was used for a 64-year-old patient with a non-resectable, but limited cholangiocarcinoma. While the course of the PHO donor was uneventful, in the DLT recipient early post-operative, dialysis-dependent renal failure with hyperoxaluria developed. Histology of a kidney biopsy revealed massive calcium oxalate crystal deposition as the leading aetiological cause. DLT using PHO organs for marginal recipients represents a possible therapeutic approach regarding graft function of the liver. However, it may negatively alter the renal outcome of the recipient in an unpredictable manner, especially with concomitant use of cyclosporin. Therefore, we suggest that, although DLT should be promoted, PHO organs are better excluded from such procedures.

Oral l-arginine supplementation ameliorates urinary risk factors and kinetic modulation of Tamm–Horsfall glycoprotein in experimental hyperoxaluric rats

Background Oral supplementation of l-arginine ( l-arg) is found to be beneficial in many kidney disorders. We determined whether l-arg supplementation safeguards the renal epithelial cell damage induced by hyperoxaluria with excretion of urinary marker enzymes and lithogenic salts with special reference to Tamm–Horsfall glycoprotein (THP). Methods Hyperoxaluria was induced by 0.75% ethylene glycol (EG) in drinking water. l-Arg was co-supplemented at the dose of 1.25 g/kg b.w. orally for 28 days. At the end of experimental period, 24-h urine samples were collected in all the experimental groups. Isolation and purification of THP was carried in rat urine and were subjected to spectrophotometric crystallization assay and calcium– 14C-oxalate binding studies. Determination of the lithogenic risk factors like calcium, oxalate, phosphorus, citrate, and marker enzymes such as lactate dehydrogenase (LDH) and γ-glutamyltransferase (γ-GT) were carried out in the collected urine sample. Results Urinary excretion of calcium and oxalate was significantly increased in EG-treated rats. In l-arg supplemented hyperoxaluric rats, these concentrations were significantly ( p < 0.001) decreased when compared to that of hyperoxaluric rats, and were moderately elevated from that of control rats. The activities of urinary marker enzymes, both LDH and γ-GT were 2-fold increased in EG-treated rats, when compared to control rats, but these values were maintained near normal in l-arg supplemented EG-treated rats. Citrate excretion was enhanced in the l-arg co-supplemented hyperoxaluric rats. In spectrophotometric crystallization assay system, l-arg supplemented rat THP showed inhibition in nucleation and aggregation phases, whereas EG-treated rat THP showed promotion of both calcium oxalate nucleation and aggregation phases. In calcium– 14C-oxalate binding assay, THP derived from hyperoxaluric rats exhibited 2-fold increase ( p < 0.001) in the Ca * Ox binding when compared to control and l-arg supplemented animals. Conclusions l-Arg could act as a potent antilithic agent, by increasing the level of citrate in the hyperoxaluria-induced rats and decreasing calcium oxalate binding to the THP. l-Arg also effectively prevents the deposition of calcium oxalate crystals by curtailing the renal epithelial damage and protein oxidation as evidenced by the normal activities of urinary marker enzymes in l-arg supplemented hyperoxaluric rats.

Vitamin E therapy prevents hyperoxaluria‐induced calcium oxalate crystal deposition in the kidney by improving renal tissue antioxidant status

To determine whether vitamin E prevents hyperoxaluria-induced stone formation, using a new animal model of calcium oxalate stone disease, as our previous in- vitro and in-vivo studies showed that oxalate and hyperoxaluria induce free-radical generation, which results in peroxidative injury to renal tubular cells. Ethylene glycol (EG) was administered at 150 mg/day by gavage for 3 weeks to rats fed on diets with adequate (group 1), excess (group 2) or deficient (group 3) vitamin E. Several indicators of peroxidation, free radicals and enzymatic activity were then assessed. EG treatment in group 1 lead to increased lipid peroxidation, protein thiol, excretion of urinary enzymes, oxalate and decreases in urinary calcium, antioxidant enzymes and altered glutathione redox balance. Although renal function was not altered, there was increased water intake, urine volume and lowered urinary pH in these rats. These changes were more intense, with extensive calcium-oxalate crystal deposition, in rats in group 3, and prevented in rats in group 2, except for urinary oxalate levels, which remained high. Histopathological examination showed that there was no deposition of calcium oxalate crystals in rats in group 2. This is the first study to demonstrate in-vivo evidence that hyperoxaluria-induced peroxidative injury induces individual calcium oxalate crystal attachment in the renal tubules. In addition, excess vitamin E completely prevented calcium oxalate deposition, by preventing peroxidative injury and restoring renal tissue antioxidants and glutathione redox balance. Therefore, vitamin E therapy might provide protection against the deposition of calcium oxalate stones in the kidney of humans.

Heavy metal stress reduces the deposition of calcium oxalate crystals in leaves of Phaseolus vulgaris

Calcium oxalate (CaOx) crystals in plants may serve as a sink for the absorption of excess calcium, and they could play an important role in heavy metal detoxification. In this study, the effect of heavy metals and different calcium concentrations on the growth of calcium oxalate crystals in leaves of Phaseolus vulgaris was investigated. Different analytical techniques were used to determine the influence of exogenous lead and zinc on CaOx deposition and to detect a presence of these metals in CaOx crystals. We found a positive correlation between the calcium concentration in the nutrient medium and the production of calcium oxalate crystals in leaves of hydroponically grown plants. On the other hand, addition of the heavy metals to the nutrient medium decreased the number of crystals. Energy dispersive X-ray spectrometry did not detect the inclusion of heavy metals inside the CaOx crystals. Our investigation suggests that CaOx crystals do not play a major role in heavy metal detoxification in P. vulgaris but do play an important role in bulk calcium regulation.

Hypocitraturia as a risk factor for nephrocalcinosis after kidney transplantation

Calcium-oxalate crystal deposition in kidney transplant biopsy specimen led us to investigate the impact of calcineurin inhibitor treatment on urinary excretion of lithogenic and stone inhibitory substances in 53 children after successful kidney transplantation (KTx) receiving cyclosporine A (CsA) or tacrolimus. We compared the values obtained with those of 12 patients with recurrent nephrotic syndrome under CsA and of 6 patients with Rasmussen encephalitis (RE) under tacrolimus therapy. Renal ultrasound examinations were repeatedly performed. Hypocitraturia was found in 69% of patients, with KTx patients having a significantly lower urinary citrate excretion than those receiving calcineurin inhibitors for other reasons. Secondly, we found hyperoxaluria in 35% of patients, again especially in those after KTx. No significant difference in urinary substances was seen comparing CsA with tacrolimus treatment. Urolithiasis was found in one and calcium-oxalate crystal deposition in biopsy specimen of three KTx patients. Calcineurin inhibitor treatment can lead to significant hypocitraturia, especially in patients after KTx receiving the highest dose of medication. Hyperoxaluria is primarily the result of a removal of significant body oxalate stores, deposited during dialysis, but may not be suspected as a specific side effect of calcineurin inhibitor therapy. Both findings can increase the risk for urolithiasis or nephrocalcinosis.

Successful transplantation of a liver from a donor with fatal ethylene glycol poisoning

Successful transplantation of a liver from a donor with fatal ethylene glycol poisoning

1124: Do Mice have a Key to Conquer Urolithiasis?- Mice may Delete Calcium Oxalate Crystal Deposits from their Kidney with Osteopontin

You have accessJournal of UrologyDiscussed Poster, Monday, May 10, 2004, Monday, May 10, 2004 1:00 - 5:00 pm1 Apr 20041124: Do Mice have a Key to Conquer Urolithiasis?- Mice may Delete Calcium Oxalate Crystal Deposits from their Kidney with Osteopontin Atsushi Okada, Bing Gao, Sumio Endo, Masahito Hirose, Mugi Yoshimura, Yasunori Itoh, Takahiro Yasui, Keiichi Tozawa, and Kenjiro Kohri Atsushi OkadaAtsushi Okada More articles by this author , Bing GaoBing Gao More articles by this author , Sumio EndoSumio Endo More articles by this author , Masahito HiroseMasahito Hirose More articles by this author , Mugi YoshimuraMugi Yoshimura More articles by this author , Yasunori ItohYasunori Itoh More articles by this author , Takahiro YasuiTakahiro Yasui More articles by this author , Keiichi TozawaKeiichi Tozawa More articles by this author , and Kenjiro KohriKenjiro Kohri More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)38361-7AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1124: Do Mice have a Key to Conquer Urolithiasis?- Mice may Delete Calcium Oxalate Crystal Deposits from their Kidney with Osteopontin." The Journal of Urology, 171(4S), p. 296 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 171Issue 4SApril 2004Page: 296 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Atsushi Okada More articles by this author Bing Gao More articles by this author Sumio Endo More articles by this author Masahito Hirose More articles by this author Mugi Yoshimura More articles by this author Yasunori Itoh More articles by this author Takahiro Yasui More articles by this author Keiichi Tozawa More articles by this author Kenjiro Kohri More articles by this author Expand All Advertisement PDF downloadLoading ...

Oxalose

Oxalosis is the systemic deposition of calcium oxalate crystals, caused by primary hyperoxaluria type 1 (PH1), the most frequent variety of primary hyperoxaluria. PH1 is an autosomal recessive disease due to an hepatic enzymatic defect of alanine : glyoxylate aminotransferase (AGT), resulting in oxalate overproduction. Because of the poor solubility of calcium oxalate in urine, the first symptom is usually a recurrent lithiasis, beginning in childhood. Associated nephrocalcinosis results almost always in terminal renal failure before the adult age, and oxalate crystals accumulate in many tissues, particularly bones. Liver transplantation, or more often combined liver-kidney transplantation, is the only curative treatment ; at an early stage, permanent urine dilution by hyperhydration may prevent nephrocalcinosis. Primary hyperoxaluria type 2, a much rarer disease due to a defect of glyoxylate/hydroxypyruvate-reductase (gr/HPR), is associated with L-glyceric aciduria. Its main manifestation is also recurrent lithiasis ; however renal failure is a rare occurrence.

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

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. 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. 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. 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.

Renal calcium phosphate and oxalate deposition in prolonged vitamin B6 deficiency: studies on a rat model of urolithiasis.

To determine the effect on kidney oxalate-salt deposition of a prolonged diet which induced vitamin B6 deficiency in adult rats, as there are reports of the pathogenic involvement of vitamin B6 deficiency in the formation of renal calcium oxalate calculi. The study comprised 24 6-month-old male albino Wistar rats; 12 were fed with a purified vitamin B6-deficient diet and the others provided with the same diet but supplemented with 6 mg/kg of vitamin B6. After 12 weeks, all rats were killed, and their kidneys fixed in formalin and routinely processed to paraffin for morphological examination; some fragments were fixed in glutaraldehyde and prepared for ultrastructural examination. From each rat consecutive sections of both kidneys were cut and stained with haematoxylin and eosin, periodic-acid Schiff, Sirius red and the Von Kossa method for calcium. Sections were examined in polarized light and by electron microscopy. The histopathological and ultrastructural features of the kidney of vitamin B6-deficient rats were those of tubular-interstitial nephritis, characterized by tubular atrophy, interstitial fibrosis and chronic inflammatory infiltration. Oxalate and phosphate crystals were present in the papillary and parenchymal connective tissue. Ultrastructural features confirmed severe tubular epithelial lesions and the presence of an interstitial and intraepithelial inflammatory infiltrate; there was mild interstitial fibrosis. None of these features were apparent in the kidney of control rats. Histopathological and ultrastructural data indicate that a prolonged vitamin B6-deficient diet may contribute to the formation and deposition of calcium phosphate and oxalate crystals, which lead to severe damage of the renal parenchyma. This phenomenon may occur not only in growing rats, which have more active protein metabolism and consequently higher vitamin B6 requirements, but also in adult rats.

Calcium oxalate stone disease: role of lipid peroxidation and antioxidants.

Membrane injury facilitated the fixation of calcium oxalate crystals and subsequent growth into kidney stones. Oxalate-induced membrane injury was mediated by lipid peroxidation reaction through the generation of oxygen free radicals. In urolithic rat kidney or oxalate exposed cultured cells, both superoxide anion and hydroxyl radicals were generated in excess, causing cellular injury. In hyperoxaluric rat kidney, both superoxide and H2O2-generating enzymes such as glycolic acid oxidase (GAO) and xanthine oxidase (XO) were increased, and hydroxyl radical and transition metal ions, iron, and copper were accumulated. The lipid peroxidation products, thiobarbituric acid-reactive substances (TBARS), hydroperoxides, and diene conjugates were excessively released in tissues of urolithic rats and in plasma of rats as well as stone patients. The accumulation of these products was concomitant with the decrease in the antioxidant enzymes, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glucose-6 phosphate dehydrogenase (G6PD) as well as radical scavengers, vitamin E, ascorbic acid, reduced glutathione (GSH), and protein thiol. All the above parameters were decreased in urolithic condition, irrespective of the agents used for the induction of urolithiasis. Oxalate binding activity and calcium oxalate crystal deposition were markedly pronounced, along with decreased adenosine triphosphatase (ATPase) activity. Lipid peroxidation positively correlated with cellular oxalate, oxalate binding, gamma-glutamyl carboxylase, and calcium level and negatively correlated with GSH, vitamin E. ascorbic acid, and total protein thiol. Antioxidant therapy to urolithic rats with vitamin E, glutathione monoester, methionine, lipoic acid, or fish oil normalised the cellular antioxidant system, enzymes and scavengers, and interrupted membrane lipid and protein peroxidation reaction, ATPase inactivation, and its associated calcium accumulation. Antioxidant therapy prevented calcium oxalate precipitation in the rat kidney and reduced oxalate excretion in stone patients. Similarly, calcium oxalate crystal deposition in vitro to urothelium was prevented by free radical scavengers such as phytic acid and mannitol by protecting the membrane from free radical-mediated damage. All these observations were suggestive of the active involvement of free radical-mediated lipid peroxidation-induced membrane damage in the pathogenesis of calcium oxalate crystal deposition and retention.

Cytokine production induced by binding and processing of calcium oxalate crystals in cultured macrophages

Deposition of calcium oxalate (CaOx) crystals in the renal interstitium is common in humans with primary oxalosis and secondary hyperoxaluria, as well as in kidneys of rats with CaOx nephrolithiasis. In vivo, macrophages and multinucleated giant cells mostly encapsulate these crystals. To investigate whether macrophages are able to dispose of CaOx crystals after phagocytosis, we used a nontransformed macrophage cell line derived from mouse spleen progenitors. Cytokine assays showed that in response to crystal binding and phagocytosis, these macrophages release tumor necrosis factor-[alpha ]. This release was evident at 8 hours, maximal at 24 hours, and decreased to control values after 48 hours of incubation with crystals. A very low but significant release of interleukin-6 into the culture medium was only noticed after 32 hours. Radiochemical experiments showed that these cells bind 38.8% of the CaOx crystals added. After 4 days, all internalized crystals had been dissolved and their molecular constituents released into the extracellular environment. Confocal laser scanning microscopy followed by morphometrical analyses confirmed these results. Long-term (survival) analyses showed that in the interval under study and at the crystal doses used, cell viability was not significantly affected. These findings support the view that properly functioning macrophages are able to remove CaOx deposits from the renal interstitium and that these cells produce inflammatory cytokines before crystal dissolution. [copy ] 2001 by the National Kidney Foundation, Inc.

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

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. 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. 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. 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.

Ultrastructure of selected calcium oxalate-containing urinary calculi from dogs.

To elucidate the ultrastructural details of calcium oxalate-containing urinary calculi from dogs. Sample Population-38 specimens selected from a collection of 8,297 oxalate-containing urinary calculi from dogs: 22 specimens composed of calcium oxalate (calcium oxalate monohydrate [COM], calcium oxalate dihydrate [COD], or COM and COD) and 16 specimens composed of calcium oxalate with amorphous calcium phosphate. Analyses of specimens included use of plain, reflected, and polarized light microscopy, X-ray diffractometry, scanning electron microscopy (SEM) with backscattered electron (BSE) imagery, and electron microprobe analysis. Four texture types were observed in calcium oxalate calculi; 4 texture types of calcium oxalate-calcium phosphate-mixed calculi were recognized. Texture types were delineated through differences in calcium oxalate crystal sizes, which were affected by urine supersaturation and abundance of crystal nucleation sites. Segregation of calcium oxalate from calcium phosphate indicated they do not precipitate under the same conditions. Deposition of calcium phosphate between calcium oxalate crystals decreased the volume of pore spaces within calculi. Porosity was observed along boundaries between COM and COD. Minute pores increased the surface area of calculi exposed to urine, and this increase in liquid-solid interface promotes interaction of crystals with the surrounding urine. Calcium oxalate urolithiasis is of major concern, because it is often a recurrent disease among dogs, principally treated by surgical removal of calculi, with few effective dissolution strategies. Understanding the ultrastructure and mineralogic content of calcium oxalate and its association with amorphous calcium phosphate is a step toward the solution of this increasingly important medical problem.

Calcium oxalate microdeposition in failing kidney grafts

Forty allograft nephrectomies (28 males and 12 females, mean age of 33.2 years), performed between 1986 and 1999, were selected. Twenty patients had been grafted from cadaveric donors; of the remaining cases, 15 had received a kidney from a living related donor. Except for six patients who received steroid-azathioprine immunosuppression, all recipients were treated with cyclosporine A and low doses of steroids. None had primary or acquired hyperoxaluria as the cause of original end-stage renal disease, and none had a history of kidney stones or urinary infection. Kidney failure occurred after a time period ranging from 2 days to 11 years posttransplant and was secondary to acute rejection in 17 cases, chronic rejection in 15, ischemic necrosis in five, acute or chronic pielonephritis in two, and allergic microangiopathy in one. No blood chemistry evaluations for oxalemia were performed. Clinical data are summarized in Table 1. Multiple sections cut from formalin-fixed, paraffin-embedded blocks and stained with haematoxylin-eosin (H&E) were examined using partially crossed polarizing screens. The presence of calcium oxalate crystals in different segments of the nephron or in the interstitium was recorded. Birefringent deposits with characteristic green iridescence, presenting as large plate-like or diamondshaped crystals, or any shape, provided that the crystals displaced tissue structures, were studied. Confirmation of the crystal nature was obtained using Yasue’s method, a histochemical stain considered to be specific for calcium oxalate, while usual stains for calcium (alizarin red and von Kossa’s method) were negative. Silver nitrate–rubeanic acid with 5% acetic acid pretreatment (Yasue’s method) stains only calcium oxalate, since calcium phosphate and calcium carbonate are dissolved with the acetic acid. A thyroid with multiple deposits of calcium oxalate and hydroxyapatite crystals was used as a positive control for the histochemical stains.

Interaction between Osteopontin on Madin Darby Canine Kidney Cell Membrane and Calcium Oxalate Crystal

We recently reported that the addition of the protein osteopontin (OPN) resulted in an increase in the deposition of calcium oxalate (CaOx) crystals on the surface of Madin Darby canine kidney (MDCK) cells. To determine the degree to which this increased deposition is caused by OPN, we investigated the extent to which the CaOx crystal deposition produced by the expression of OPN at the cell surface was suppressed by 4 different methods prior to the determination of the level of CaOx crystal binding. MDCK cells (2 × 10⁶ cells/well) were cultured to a confluent state, and the binding of OPN to the cellular surface was then inhibited by adding one of the following 4 substances: human OPN polyclonal antibody, thrombin, cyclic Arg-Gly-Asp (RGD) peptides and tunicamycin. The cells were cultured for 24 h. We then used a fluorescent antibody technique with an OPN polyclonal antibody to determined whether the expression of OPN at the cell surface was inhibited, and we measured the degree of CaOx crystal deposition using the isotope ⁴⁵Ca. The degree of CaOx crystal deposition was inhibited by 80% or more in the antibody-treated group, by 50–80% in the thrombin-treated group, by 60–80% in the cyclic RGD-treated group, and by 50–60% in the tunicamycin-treated group. These results suggest that OPN in the extracellular matrix is the main cause of CaOx crystal deposition on the surface of MDCK cells.