Calcium Oxalate Crystal Retention Research Articles

Oxalate Nephropathy.

Oxalate Nephropathy.

Anecdotes of lithogenesis and atherogenesis conversely liable for cardiac dysfunction and kidney stone formation

Dear Editor, Recently, Masterson et al. [1] reported that dyslipidemia increases the risk of kidney stone formation. Interestingly, in 2002 Khan et al. reported [2] that urolithic patients excrete more lipids such as total cholesterol, triglycerides, and some phospholipids [2]. The same study further suggested that lipids modulate the calcium oxalate crystal nucleation, aggregation and retention processes, which are the important steps for kidney stone development [2]. On the other hand, the association of kidney stones and subclinical carotid atherosclerosis has been observed in young adults implying that renal lithogenesis and atherogenesis share common pathophysiology both systemically and on a cellular level [3], where oxalate would act as a pro-atherogenic factor to increase the oxidative stress [2] as seen in atherosclerosis patients [3]. Injury and retention model explains the lithogenesis and atherogenesis [2, 3]. In kidney stones, retention of calcium oxalate crystals occurs in the injured urothelium, inducing accumulation of lipids, proteinaceous materials, such as calcium-binding protein and glycoproteins, which interact with crystals and modulate the nucleation and aggregation process leading to stone formation [1]. Vascular cell injury and retention of lipids in the arterial wall cause the atherosclerosis [3]. In humans, all these processes take place over a long period of time [2, 3] during which several metabolic changes occur finally ending in lithogenesis and switching on atherogenesis, or vice versa. Thus, even though patients have either kidney stone alone or cardiovascular disease alone, it would be ideal for physicians to screen for risk factors of both diseases periodically. Metabolic changes can also be altered due to transient food consumption. For example, it is well accepted that oxalate and purine rich foods can lead to hyperoxaluria and hyperuricosuria, respectively, while oil rich food increases plasma cholesterol and triglycerides levels. Hence, to avoid a false diagnosis, it would be better to measure within different time frames. Further, identification of drugs inhibiting both atherogenesis and lithogenesis would enable the medical community to ease the pain of patients and be economically conservative.

Preliminary Observations of Urinary Calcium and Osteopontin Excretion in Premature Infants, Term Infants and Adults

Osteopontin is an acidic glycoprotein which may prevent nephrocalcinosis and nephrolithiasis by inhibiting the growth and retention of calcium oxalate crystals within the tubular lumen. The purpose of this study was to obtain preliminary data regarding urinary osteopontin in premature infants at risk for nephrocalcinosis. We examined urinary osteopontin concentration in premature infants, term infants and adults, and examined the relationship between urinary calcium and osteopontin concentration in these groups. The urinary osteopontin concentration of 17 premature infants of 3.7 ± 1.2 µg/ml was not significantly different from the urinary osteopontin concentration of 12 term infants of 6 ± 6 µg/ml, while the urinary osteopontin concentration in 23 urine specimens from adults of 27 ± 15 µg/ml was significantly higher than premature infants and term infants (p < 0.05). Urinary osteopontin concentration did not correlate with urinary calcium concentration in premature infants, while there was a correlation between the osteopontin/creatinine ratio and calcium/creatinine ratios in premature infants. Diminished urinary concentration of osteopontin may enhance the risk for nephrocalcinosis in premature infants.

Oxalate mediated nephronal impairment and its inhibition by c-phycocyanin: A study on urolithic rats

The assumption of oxidative stress as a mechanism in oxalate induced renal damage suggests that antioxidants might play a beneficial role against oxalate toxicity. An in vivo model was used to investigate the effect of C-phycocyanin (from aquatic micro algae; Spirulina spp.), a known antioxidant, against calcium oxalate urolithiasis. Hyperoxaluria was induced in two of the 4 groups of Wistar albino rats (n = 6 in each) by intraperitoneally injecting sodium oxalate (70 mg/kg body weight). A pretreatment of phycocyanin (100 mg/kg body weight) as a single oral dosage was given, one hour prior to oxalate challenge. An untreated control and drug control (phycocyanin alone) were employed. Phycocyanin administration resulted in a significant improvement (p < 0.001) in the thiol content of renal tissue and RBC lysate via increasing glutathione and reducing malondialdehyde levels in the plasma of oxalate induced rats (p < 0.001), indicating phycocyanin's antioxidant effect on oxalate mediated oxidative stress. Administering phycocyanin after oxalate treatment significantly increased catalase and glucose-6-phosphate dehydrogenase activity (p < 0.001) in RBC lysate suggesting phycocyanin as a free radical quencher. Assessing calcium oxalate crystal retention in renal tissue using polarization microscopy and renal ultrastructure by electron microscopy reveals normal features in phycocyanin-- pretreated groups. Thus the study presents positive pharmacological implications of phycocyanin against oxalate mediated nephronal impairment and warrants further work to tap this potential aquatic resource for its medicinal application.

Counteraction of oxalate induced nitrosative stress by supplementation of l-arginine, a potent antilithic agent

Background Our understanding of nitrosative stress in the process of urolithiasis is far from complete. Earlier studies carried out in our laboratory demonstrate the presence of nitrated THP in stone formers, l-arginine ( l-arg) a precursor of nitric oxide (NO), attenuates the endothelial dysfunction caused by reactive nitrogen species. We investigated the role of l-arg in ethylene glycol (EG)-induced urolithic rat model and observed its antilithic and antioxidative properties. Methods Hyperoxaluria was induced using 0.75% EG in drinking water. l-arg [1.25 g/kg body weight] was given orally for a period of 28 days. Results EG-treated rats showed significant loss in body weight and increase in the activities of oxalate synthesizing enzymes such as glycollic acid oxidase in liver. Lactate dehydrogenase activity in liver and kidney was increased. The activity of the free radical producing enzyme xanthine oxidase, tissue oxalate and calcium levels were significantly increased in EG-treated rats. Depletion in the antioxidant enzymes, membrane bound ATPases and thiol status was observed in these rats. l-arg co-supplementation to EG-treated rats maintained the activities of the oxalate synthesizing enzymes and free radical producing enzymes with in the normal range. Tissue oxalate and calcium levels were also maintained near normal in l-arg treated hyperoxaluric rats. l-arg, by its cytoprotective effect, maintained the thiol status, thereby preserving the activities of the membrane bound ATPases and preventing proteinuria and subsequent weight loss in EG-treated rats. Conclusion l-arg feeding prevents the retention of calcium oxalate crystals in hyperoxaluric rats by way of protecting the renal cells from oxidative injury and also by providing a second line of defense through the normalization of the oxalate metabolism. It reduces the risk of stone formation, by curtailing free radicals and hyperoxaluria as both of them have to work in close association to form stones.

Presence of lipids in urine, crystals and stones: Implications for the formation of kidney stones

Cell membranes and their lipids play critical roles in calcification. Specific membrane phospholipids promote the formation of calcium phosphate and become a part of the organic matrix of growing calcification. We propose that membrane lipids also promote the formation of calcium oxalate (CaOx) and calcium phosphate (CaP) containing kidney stones, and become a part of their stone matrix. Human urine, crystals of CaOx and CaP produced in the urine of healthy individuals, and urinary stones containing struvite, uric acid, CaOx and CaP crystals for the presence of membrane lipids were analyzed. Crystallization of CaOx monohydrate at Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) and dimyristoylphosphatidylglycerol (DMPG) was investigated to directly demonstrate that phospholipid assemblies can catalyze CaOx nucleation. Urine as well as CaOx and CaP crystals made in the urine and various types of urinary stones investigated contained some lipids. Urine of both CaOx and uric acid stone formers contained significantly more cholesterol, cholesterol ester and triglycerides than urine of healthy subjects. However, urine of CaOx stone formers contained more acidic phospholipids. The organic matrix of calcific stones contained significantly more acidic and complexed phospholipids than uric acid and struvite stones. For each Langmuir monolayer precipitation was heterogeneous and selective with respect to the orientation and morphology of the CaOx crystals. Crystals were predominantly monohydrate, and most often grew singly with the calcium rich (10-1) face toward the monolayer. The number of crystals/mm2 decreased in the order DPPG> DPPC and was inversely proportional to surface pressure and mean molecular area/molecule. Stone forming conditions in the kidneys greatly impact their epithelial cells producing significant differences in the urinary lipids between healthy and stone forming individuals. Altered membrane lipids promote face selective nucleation and retention of calcium oxalate crystals, and in the process become a part of the growing crystals and stones.

Urinary Crystallization Inhibitors Do Not Prevent Crystal Binding

Renal stone formation requires the persistent retention of crystals in the kidney. Calcium oxalate monohydrate (COM) crystal binding to Madin Darby canine kidney strain I (MDCK-I), a cell line that resembles the epithelium in the renal distal tubule/collecting duct, is developmentally regulated, while LLC-PK1 cells (American Type Tissue Collection), which are widely used as a model of the renal proximal tubule, bind crystals irrespective of their stage of epithelial development. Whereas to our knowledge the binding molecules for COM at the surface of LLC-PK1 cells are still unknown, crystals adhere to the hyaluronan (HA) rich pericellular matrix transiently expressed by mobile MDCK-I cells. In the current study we investigated whether crystal binding to either cell type is influenced by urinary substances, including glycoprotein inhibitors of crystallization We studied crystal binding to MDCK-I cells during wound repair, to confluent LLC-PK1 cells and to HA immobilized on a solid surface using [14C] COM pretreated or not pretreated with urine from healthy male volunteers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed to assess whether the crystals became coated with urine derived proteins Western blot analysis demonstrated that pretreated COM crystals were covered with protein inhibitors of crystallization. However, this protein coat had no significant effect on the level of crystal binding to either cell type. In contrast, the adherence of urine treated crystals to immobilized HA was significantly reduced The adherence of crystals to pericellular matrixes may encompass more than their simple fixation to the polysaccharide HA. Calcium oxalate crystal retention is not prevented by coating crystals with urinary constituents such as glycoproteins and, therefore, may predominantly depend on the surface properties of the renal tubular epithelium.

Effects of Tamm-Horsfall Protein and Albumin on the Inhibition of Free Radicals

Introduction: Oxalate in urine can cause tubular cellular damage by the production of free radicals. Then, cell death and cellular debris may promote the retention of calcium oxalate crystals and finally the formation of stones. The two most abundant urinary proteins, Tamm-Horsfall protein (THP) and albumin, were tested for the effects of antioxidants. Materials and Methods: By using xanthine-xanthine oxidase reaction, purified THP and albumin were tested for the inhibitory effect. OD₂₉₅ was used as a spectrophotometric method to measure the production of uric acid during the reaction. Results and Conclusions: Both proteins can inhibit the reaction of xanthine oxidase on xanthine, although the effect was decreased after enzymatic deglycosylation of sialic acid. Albumin has an IC₅₀ of 10.7 nM in native condition and 11.9 nM after deglycosylation, whereas THP has 69.6 nM in native condition and 102.0 nM in deglycosylated condition. The data indicates that THP and albumin have an antioxidant effect. Sialic acid in THP has partly an inhibitory effect and is associated with calcium oxalate formation. Studies have indicated that further investigation of the role of free radicals in the formation of urolithiasis and of sialic acid in protein function is needed.

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.

Effect of experimental hyperoxaluria on renal calcium oxalate monohydrate binding proteins in the rat.

To determine the functional role of calcium oxalate binding proteins in the nucleation, aggregation and retention of calcium oxalate crystals under physiological and hyperoxaluric conditions. Materials and methods Hyperoxaluria was induced in rats using 0.75% of ethylene glycol in drinking water. Calcium oxalate binding proteins were isolated and fractionated by cellulose column chromatography. Three major protein peak fractions were obtained (73 kDa in Tris-HCl buffer, 20 kDa in 0.05 mol/L NaCl buffer and 23 kDa in 0.3 mol/L buffer). Oxalate binding and the inhibition of crystal nucleation and aggregation by these fractions were determined. The adsorption of calcium oxalate monohydrate (COM) was ubiquitous in rat tissues and subcellular organelles, but the percentage adsorption varied; maximum absorption occurred in kidneys and pancreas, with microsomes showing maximal adsorption in the kidney. Hyperoxaluric rat tissues showed a greater percentage adsorption. Microsomes were enriched with the 20 kDa protein, while nuclei contained the 23 kDa protein in higher concentrations. COM-binding proteins derived from hyperoxaluric rat kidney had a greater content of 74 kDa and 23 kDa proteins with increased oxalate-binding activities. In the crystal-growth studies, the 74 kDa protein was a promoter, while the other protein fractions inhibited crystallization. In hyperoxaluria, the crystal-growth promoting activity of the 74 kDa protein was further increased, while the inhibition by the 20 and 23 kDa proteins was decreased. The 74 kDa protein derived from control rats formed single COM crystals in a crystal growth system, while the hyperoxaluric rat fraction induced the aggregation of COM crystals. COM-binding proteins (the 74 and 23 kDa fractions) were expressed more in hyperoxaluric rats. In hyperoxaluria the 74 kDa protein tended to promote crystal nucleation and aggregation, and the 20 and 23 kDa proteins were less inhibitory, which increases the risk of stone formation.

Effect of Oral Supplementation of Vitamin E on Urinary Risk Factors in Patients with Hyperoxaluria.

The effects of administration of vitamin E on hyperoxaluria, hypercalciuria, hyperuricosuria, and hypocitraturia and on the decreased levels of antioxidants such as ascorbic acid, vitamin E, glutathione (GSH), and superoxide dismutase (SOD) in kidney stone patients were observed. Following the surgical removal of kidney stones from the patients, vitamin E (200mg/day) was administered from the 7th day onwards to twenty patients for up to 90 days. The normalization process of urinary risk factors and antioxidants was rapid in the vitamin E-administered patients when compared with that in the untreated group. The increased excretion of citrate following vitamin E administration was suggested to reduce the retention of calcium oxalate crystals and thereby lower the rate of recurrence of stones.

Crystal Retention by Injured Urothelium of the Rat Urinary Bladder

Injuries caused by hydrochloric acid or Triton X 100 application to the rat bladder urothelium, and the effects on calcium oxalate crystal retention, were examined by light and scanning electron microscopy. Damage due to either compound resulted in desquamation of urothelial cells. The crystals appeared to be retained by a fibrillar material, some of which was identified as fibrin. Heparin treatment of injured urothelium was found to prevent crystal retention.