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Background: Calcium oxalate monohydrate (COM) forms the most common type of kidney stones observed in clinics, elevated levels of urinary oxalate being the principal risk factor for such an etiology. The objective of the present study was to evaluate the anti-nephrolithiatic effect of herbo-mineral formulation, Lithom., Methods: The in vitro biochemical synthesis of COM crystals in the presence of Lithom was performed and observations were made by microscopy and Scanning Electron Microscope (SEM) based analysis for the detection of crystal size and morphology. The phytochemical composition of Lithom was evaluated by Ultra-High-Performance Liquid Chromatography (UHPLC). The in vivo model of Ethylene glycol-induced hyperoxaluria in Sprague-Dawley rats was used for the evaluation of Lithom. The animals were randomly allocated to 5 different groups namely Normal control, Disease control (ethylene glycol (EG), 0.75%, 28 days), Allopurinol (50 mg/kg, q.d. ), Lithom (43 mg/kg, b.i.d. ), and Lithom (129 mg/kg, b.i.d. ). Analysis of crystalluria, oxalate, and citrate levels, oxidative stress parameters (malondialdehyde (MDA), catalase, myeloperoxidase (MPO)), and histopathology by hematoxylin and eosin (H&E) and Von Kossa staining was performed for evaluation of Lithom., Results: The presence of Lithom during COM crystals synthesis significantly reduced the average crystal area, feret's diameter, and area-perimeter ratio, in a dose-dependent manner. SEM analysis revealed that COM crystals synthesized in the presence of 100 and 300 μg/mL of Lithom exhibited a veritable morphological transition from irregular polygons with sharp edges to smoothened smaller cuboid polygons. UHPLC analysis of Lithom revealed the presence of Trigonelline, Bergenin, Xanthosine, Adenosine, Bohoervinone B, Vanillic acid, and Ellagic acid as key phytoconstituents. In EG-induced SD rats, the Lithom-treated group showed a decrease in elevated urinary oxalate levels, oxidative stress, and renal inflammation. Von Kossa staining of kidney tissue also exhibited a marked reduction in crystal depositions in Lithom-treated groups., Conclusion: Taken together, Lithom could be a potential clinical-therapeutic alternative for management of nephrolithiasis.

Short bowel (SB) increases the risk of kidney stones. However, the underlying mechanism is unclear. Here, we examined how SB affected renal oxalate and citrate handlings for in vivo hyperoxaluric rats and in vitro tubular cells. SB was induced by small intestine resection in male Wistar rats. Sham-operated controls had no resection. After 7 days of recovery, the rats were divided into control, SB (both fed with distilled water), ethylene glycol (EG), and SB+EG (both fed with 0.75% EG for hyperoxaluric induction) groups for 28 days. We collected the plasma, 24 h of urine, kidney, and intestine tissues for analysis. Hypocitraturia was found and persisted up to 28 days for the SB group. Hypocalcemia and high plasma parathyroid hormone (PTH) levels were found in the 28-day SB rats. SB aggravated EG-mediated oxalate nephropathy by fostering hyperoxaluria and hypocitraturia, and increasing the degree of supersaturation and calcium oxalate (CaOx) crystal deposition. These effects were associated with renal up-regulations of the oxalate transporter solute carrier family 26 (Slc26)a6 and citrate transporter sodium-dependent dicarboxylate cotransporter-1 (NaDC-1) but not Slc26a2. The effects of PTH on the SB kidneys were then examined in NRK-52E tubular cells. Recombinant PTH attenuated oxalate-mediated cell injury and up-regulated NaDC-1 via protein kinase A (PKA) activation. PTH, however, showed no additive effects on oxalate-induced Slc26a6 and NaDC-1 up-regulation. Together, these results demonstrated that renal NaDC-1 upregulation-induced hypocitraturia weakened the defense against Slc26a6-mediated hyperoxaluria in SB kidneys for excess CaOx crystal formation. Increased tubular NaDC-1 expression caused by SB relied on PTH., (© 2020 The Author(s).)

Calcium oxalate (CaOx) crystal deposition within the tubules is often a perplexing finding on renal biopsy of both native and transplanted kidneys. Understanding the underlying causes may help diagnosis and future management. The most frequent cause of CaOx crystal deposition within the kidney is hyperoxaluria. When this is seen in native kidney biopsy, primary hyperoxaluria must be considered and investigated further with biochemical and genetic tests. Secondary hyperoxaluria, for example due to enteric hyperoxaluria following bariatric surgery, ingested ethylene glycol or vitamin C overdose may also cause CaOx deposition in native kidneys. CaOx deposition is a frequent finding in renal transplant biopsy, often as a consequence of acute tubular necrosis and is associated with poorer long-term graft outcomes. CaOx crystal deposition in the renal transplant may also be secondary to any of the causes associated with this phenotype in the native kidney. The pathophysiology underlying CaOx deposition is complex but this histological phenotype may indicate serious underlying pathology and should always warrant further investigation.

Peroxisome proliferator-activated receptor- (PPAR-) γ is a ligand-dependent transcription factor, and it has become evident that PPAR- γ agonists have renoprotective effects, but their influence and mechanism during the development of calcium oxalate (CaOx) nephrolithiasis remain unknown. Rosiglitazone (RSG) was used as a representative PPAR- γ agonist in our experiments. The expression of transforming growth factor- β 1 (TGF- β 1), hepatocyte growth factor (HGF), c-Met, p-Met, PPAR- γ , p-PPAR- γ (Ser112), Smad2, Smad3, pSmad2/3, and Smad7 was examined in oxalate-treated Madin-Darby canine kidney (MDCK) cells and a stone-forming rat model. A CCK-8 assay was used to evaluate the effects of RSG on cell viability. In addition, intracellular reactive oxygen species (ROS) levels were monitored, and lipid peroxidation in renal tissue was detected according to superoxide dismutase and malondialdehyde levels. Moreover, the location and extent of CaOx crystal deposition were evaluated by Pizzolato staining. Our results showed that, both in vitro and in vivo , oxalate impaired PPAR- γ expression and phosphorylation, and then accumulative ROS production was observed, accompanied by enhanced TGF- β 1 and reduced HGF. These phenomena could be reversed by the addition of RSG. RSG also promoted cell viability and proliferation and decreased oxidative stress damage and CaOx crystal deposition. However, these protective effects of RSG were abrogated by the PPAR- γ -specific inhibitor GW9662. Our results revealed that the reduction of PPAR- γ activity played a critical role in oxalate-induced ROS damage and CaOx stone formation. RSG can regulate TGF- β 1 and HGF/c-Met through PPAR- γ to exert antioxidant effects against hyperoxaluria and alleviate crystal deposition. Therefore, PPAR- γ agonists may be expected to be a novel therapy for nephrolithiasis, and this effect is related to PPAR- γ -dependent suppression of oxidative stress., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Ya-Dong Liu et al.)

Interaction of pioglitazone (PGZ) and macrophages (Mps) in renal crystal formation remains unclear. In the present study, we investigated the possible mechanisms involved with Mps of PGZ in suppressing renal crystal formation. Crystal formation in the mouse kidney was detected using polarized light optical microscopy and Pizzolato staining. Gene expression was detected by Western blot analysis, quantitative RT-PCR, immunohistochemistry, immunofluorescence, and ELISA. Mp phenotypes were identified by flow cytometric analysis. Cell apoptosis was detected with TUNEL assay, and tubular injury was detected with periodic acid-Schiff staining. Interaction of peroxisome proliferator-activated receptor (PPAR)-γ and promoter was determined by chromatin immunoprecipitation assay. Luciferase reporter assay was performed to authenticate target genes of miRNA-23 (miR-23). Recombinant adenovirus was used to elucidate the role of miR-23 in vivo. Renal crystal formation, inflammation, tubular injury, and cell apoptosis were significantly marked in glyoxylic acid-treated groups and significantly decreased in PGZ-treated groups. PGZ significantly reduced Mp infiltration and M1 Mp polarization in the kidney. In vitro, PGZ shifted crystal-stimulated M1-predominant Mps to M2-predominant Mps, which were anti-inflammatory. PPAR-γ could directly bind to one PPAR-γ regulatory element in the promoter of pre-miR-23 to promote expression of miR-23 in Mps. We identified two downstream target genes of miR-23, interferon regulatory factor 1 and Pknox1. Moreover, miR-23 decreased crystal deposition, M1 Mp polarization, and injury in the kidney. This study has proven that PGZ decreased renal calcium oxalate crystal formation and renal inflammatory injury by suppressing M1 Mp polarization through a PPAR-γ-miR-23-interferon regulatory factor 1/Pknox1 axis. PGZ is liable to be a potential therapeutic medicine for treating urolithiasis.

Hyperoxaluria-induced oxidative injury of renal tubular epithelial cell is a casual and essential factor in kidney calcium oxalate (CaOx) stone formation. Autophagy has been shown to be critical for the regulation of oxidative stress-induced renal tubular injury; however, little is known about its role in kidney CaOx stone formation. In the present study, we found that the autophagy antagonist chloroquine could significantly attenuate oxalate-induced autophagy activation, oxidative injury and mitochondrial damage of renal tubular cells in vitro and in vivo, as well as hyperoxaluria-induced CaOx crystals depositions in rat kidney, whereas the autophagy agonist rapamycin exerted contrasting effects. In addition, oxalate-induced p38 phosphorylation was significantly attenuated by chloroquine pretreatment but was markedly enhanced by rapamycin pretreatment, whereas the protective effect of chloroquine on rat renal tubular cell oxidative injury was partly reversed by a p38 protein kinase activator anisomycin. Furthermore, the knockdown of Beclin1 represented similar effects to chloroquine on oxalate-induced cell oxidative injury and p38 phosphorylation in vitro. Taken together, our results revealed that autophagy inhibition could attenuate oxalate-induced oxidative injury of renal tubular cell and CaOx crystal depositions in the rat kidney via, at least in part, inhibiting the activation of p38 signaling pathway, thus representing a novel role of autophagy in the regulation of oxalate-induced renal oxidative injury and CaOx crystal depositions for the first time., (Copyright © 2018. Published by Elsevier B.V.)

Metabolic syndrome (MS) individuals have a higher risk of developing chronic kidney disease through unclear pathogenic mechanisms. MS has been also related with higher nephrolithiasis prevalence. To establish the influence of MS on renal function, we designed a murine model of combined metabolic syndrome and hyperoxaluria. Four groups of male Sprague-Dawley rats were established: (1) control group (n = 10) fed with standard chow; (2) stone former group (SF) (n = 10) fed with standard chow plus 0.75% ethylene glycol administered in the drinking water; (3) metabolic syndrome group (MS) (n = 10), fed with 60% fructose diet; (4) metabolic syndrome + stone former group (MS + SF) (n = 10), 60% fructose diet and 0.75% EG in the drinking water. MS group showed a significant injury to renal function when hyperoxaluria was induced. It was demonstrated by a significant decrease of creatinine clearance (p < 0.001), with higher tubular damage (34.3%, CI 95% 23.9-44.7, p < 0.001), produced by deposition of crystals, and increased tubular synthesis of osteopontin as a response to tubular damage. Induction of hyperoxaluria in rats with MS causes severe morphological alterations with a significant impairment of renal function. This impairment is not produced in rats without MS. Therefore, this model can be useful for the study of the influence of MS in stone formation.

In our previous report, M2-macrophage (Mφs) deficient mice showed increased renal calcium oxalate (CaOx) crystal formation; however, the role of Mφs-related-cytokines and chemokines that affect kidney stone formation remains unknown. Here, we investigated the role of M1/M2s in crystal development by using in vitro and in vivo approaches. The crystal phagocytic rate of bone marrow-derived M2Mφs was higher than that of bone marrow-derived Mφs and M1Mφs and increased on co-culture with renal tubular cells (RTCs). However, the amount of crystal attachment on RTCs reduced on co-culture with M2Mφs. In six hyperoxaluric C57BL/6J mice, M1Mφ transfusion and induction by LPS and IFN-γ facilitated renal crystal formation, whereas M2Mφ transfusion and induction by IL-4 and IL-13 suppressed renal crystal formation compared with the control. These M2Mφ treatments reduced the expression of crystal-related genes, such as osteopontin and CD44, whereas M1Mφ treatment increased the expression of pro-inflammatory and adhesion-related genes such as IL-6, inducible NOS, TNF-α, C3, and VCAM-1. The expression of M2Mφ-related genes was lower whereas that of M1Mφ-related genes was higher in papillary tissue of CaOx stone formers. Overall, our results suggest that renal crystal development is facilitated by M1Mφs, but suppressed by M2Mφs.

Introduction and Objectives: Reactive oxygen species (ROS) are produced during the interaction between oxalate/calcium oxalate monohydrate (COM) crystals and renal epithelial cells and are responsible for the various cellular responses through the activation of NADPH oxidase (Nox). Ox and COM also activate the renin-angiotensin-aldosterone system (RAAS). Aldosterone stimulates ROS production through activation of Nox with the involvement of mineralocorticoid receptor (MR), Rac1 and mitogen-activated protein kinases (MAPK). We investigated RAAS pathways in vivo in an animal model of hyperoxaluria and in vitro by exposing renal epithelial cells to COM crystals., Methods: Hyperoxaluria was induced in male SD rats by administering ethylene glycol. One group of rats was additionally given spironolactone. Total RNA was extracted and subjected to genomic microarrays to obtain global transcriptome data. Normal rat kidney cell line (NRK-52E) was incubated with aldosterone(10(-7) M) and COM(67 μg/cm(2)) with or without spironolactone(10(-5) M), a selective inhibitor of SRC family of kinases; protein phosphatase 2(pp2) (10(-5) M) and Nox inhibitor; diphenylene iodonium (DPI) (10(-5) M)., Results: Relative expression of genes encoding for AGT, angiotensin receptors 1b and 2, Renin 1, Cyp11b, HSD11B2, Nr3c2, NOx4 and Rac1 was upregulated in the kidneys of rats with hyperoxaluria. Treatment with spironolactone reversed the effect of hyperoxaluria. Both aldosterone and COM crystals activated Nox and Rac1 expression in NRK52E, while spironolactone inhibited Nox and Rac1 expression. Increased Rac1 expression was significantly attenuated by treatment with PP2 and spironolactone., Conclusions: Results indicate that hyperoxaluria-induced production of ROS, injury and inflammation are in part associated with the activation of Nox through renin-angiotensin-aldosterone pathway.

Enhanced sodium excretion is associated with intrarenal oxidative stress. The present study evaluated whether oxidative stress caused by high sodium (HS) may be involved in calcium oxalate crystal formation. Male rats were fed a sodium-depleted diet. Normal-sodium and HS diets were achieved by providing drinking water containing 0.3% and 3% NaCl, respectively. Rats were fed a sodium-depleted diet with 5% hydroxyl-L-proline (HP) for 7 and 42 days to induce hyperoxaluria and/or calcium oxalate deposition. Compared to normal sodium, HS slightly increased calcium excretion despite diuresis; however, the result did not reach statistical significance. HS did not affect the hyperoxaluria, hypocalciuria or supersaturation caused by HP; however, it increased calcium oxalate crystal deposition soon after 7 days of co-treatment. Massive calcium oxalate formation and calcium crystal excretion in HS+HP rats were seen after 42 days of treatment. HP-mediated hypocitraturia was further exacerbated by HS. Moreover, HS aggravated HP-induced renal injury and tubular damage via increased apoptosis and oxidative stress. Increased urinary malondialdehyde excretion, in situ superoxide production, NAD(P)H oxidase and xanthine oxidase expression and activity, and decreased antioxidant enzyme expression or activity in the HS+HP kidney indicated exaggerated oxidative stress. Interestingly, this redox imbalance was associated with reduced renal osteopontin and Tamm-Horsfall protein expression (via increased excretion) and sodium-dependent dicarboxylate cotransporter NaDC-1 upregulation. Collectively, our results demonstrate that a HS diet induces massive crystal formation in the hyperoxaluric kidney; this is not due to increased urinary calcium excretion but is related to oxidative injury and loss of anticrystallization defense.

Purpose: We investigated the association of hyperoxaluria and calcium oxalate crystal induced production of reactive oxygen species with activation of the NLRP3 inflammasome., Materials and Methods: Eight-week-old male rats were given hydroxy-L-proline to induce hyperoxaluria. A group of rats on the hydroxy-L-proline diet also received apocynin, an antioxidant and nonspecific inhibitor of NADPH oxidase. At 28 days the rats were sacrificed and the kidneys were extracted. Microarray analysis was done with the BeadArray™ Reader. Gene ontology and gene pathway analyses were done with the DAVID (Database for Annotation, Visualization of Integrated Discovery) enrichment analysis tool. Quantitative real-time polymerase chain reaction and immunohistochemical staining were performed to confirm microarray results., Results: Analysis of 22,226 genes revealed that 20 and 24 pathways were highly significant in the cortex and medulla, respectively. In the cortex extracellular matrix-receptor interaction, complement and coagulation cascades, focal adhesion and hypertrophic cardiomyopathy were the most significant pathways. In the medulla complement and coagulation cascades, extracellular matrix-receptor interaction and dilated cardiomyopathy were the major pathways. Genes encoding for PYCARD (ASC), TXNIP, NLRP3, caspase-1, and IL-1β and 18 were significantly up-regulated in hydroxy-L-proline fed rats but in the group that received apocynin these genes were down-regulated in the cortex and medulla. Results were verified by quantitative real-time polymerase chain reaction with SYBR® Green assay and immunohistochemical staining., Conclusions: Results indicate a role for reactive oxygen species in activation of the NLRP3 inflammasome via TXNIP. This led to a robust inflammatory response in the kidneys of rats with hyperoxaluria and calcium oxalate nephrolithiasis., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objectives: To experimentally evaluate the clinical application of N-methyl-4-isoleucine cyclosporin, a novel selective inhibitor of cyclophilin D activation., Methods: In vitro, cultured renal tubular cells were exposed to calcium oxalate monohydrate crystals and treated with N-methyl-4-isoleucine cyclosporin. The mitochondrial membrane was stained with tetramethylrhodamine ethyl ester perchlorate and observed. In vivo, Sprague-Dawley rats were divided into four groups: a control group, an ethylene glycol group (administration of ethylene glycol to induce renal calcium crystallization), a N-methyl-4-isoleucine cyclosporin group (administration of N-methyl-4-isoleucine cyclosporin) and an ethylene glycol + N-methyl-4-isoleucine cyclosporin group (administration of ethylene glycol and N-methyl-4-isoleucine cyclosporin). Renal calcium crystallization was evaluated using Pizzolato staining. Oxidative stress was evaluated using superoxide dismutase and 8-hydroxy-deoxyguanosine. Mitochondria within renal tubular cells were observed by transmission electron microscopy. Cell apoptosis was evaluated using cleaved caspase-3., Results: In vitro, calcium oxalate monohydrate crystals induced depolarization of the mitochondrial membrane potential, which was remarkably prevented by N-methyl-4-isoleucine cyclosporin. In vivo, ethylene glycol administration induced renal calcium crystallization, oxidative stress, mitochondrial collapse and cell apoptosis in rats, which were significantly prevented by N-methyl-4-isoleucine cyclosporin., Conclusions: Herein we first report a new treatment agent determining renal calcium crystallization through cyclophilin D activation., (© 2014 The Japanese Urological Association.)

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.

Objectives: Polymorphism of the gene for matrix GLA protein (MGP), a calcification inhibitor, is associated with nephrolithiasis. However, experimental investigations of MGP role in stone pathogenesis are limited. We determined the effect of renal epithelial exposure to oxalate (Ox), calcium oxalate (CaOx) monohydrate (COM) or hydroxyapatite (HA) crystal on the expression of MGP., Methods: MDCK cells in culture were exposed to 0.3, 0.5 or 1 mM Ox and 33, 66 or 133-150 μg/cm(2) of COM/HA for 3-72 h. MGP expression and production were determined by Western blotting and densitometric analysis. Enzyme-linked immunosorbent assay was performed to determine MGP release into the medium. Hyperoxaluria was induced in male Sprague-Dawley rats by feeding hydroxyl-L-proline. Immunohistochemistry was performed to detect renal MGP expression., Results: Exposure to Ox and crystals led to time- and concentration-dependent increase in expression of MGP in MDCK cells. Cellular response was quicker to crystal exposure than to the Ox, expression being significantly higher after 3-h exposure to COM or HA crystals and more than 6 h of exposure to Ox. MGP expression was increased in kidneys of hyperoxaluric rats particularly in renal peritubular vessels., Conclusion: We demonstrate increased expression of MGP in renal tubular epithelial cells exposed to Ox or CaOx crystals as well as the HA crystals. The most significant finding of this study is the increased staining seen in renal peritubular vessels of the hyperoxaluric rats, indicating involvement of renal endothelial cells in the synthesis of MGP.

Background and Purpose: We previously reported hyperoxaluria and calcium oxalate calculi in adult pigs (sows) fed hydroxyproline (HP). The purpose of this study was to grossly and histopathologically characterize intrarenal effects in this model., Methods: In the swine facility at our campus, we maintained 21 gestating sows, of which 15 received daily treatment (5% HP mixed with dry feed) and 6 received no treatment (controls). Nine were sacrificed at 21 d (three control, six HP). All kidneys were extracted and examined grossly and for radiographic evidence of stones (GE CT scanner, 80kV, 400MA, 1 sec rotation, 0.625 mm slices). Papillary and cortical samples were processed for histologic analysis., Results: Kidneys from treated sows showed significant calculi distributed within the renal papilla on CT, appeared mottled in the renal cortex and papillary areas, and had less distinct corticomedullary borders. Tiny crystals and mucinous debris lined the papillary tips, calices, and pelvis in kidneys from four of six treated sows, and multiple stones were noted at the papillary tips. Hematoxylin and eosin stain revealed crystals in collecting tubules and papillary tips in treated kidneys and none in controls. Yasue staining confirmed crystals in proximal periglomerular tubules of treated but not control animals. Tubular dilation and inflammatory/fibrotic changes were identified in kidneys from treated animals; none of these changes were evident in control kidneys., Conclusions: We report renal damage as a result of dietary-induced hyperoxaluria in adult sows. Specifically, we found crystalluria in proximal periglomerular tubules and collecting ducts, with tubular damage at all segments.

Purpose: To compare renal function and metabolic abnormalities of cystine stone patients and calcium oxalate stone patients in China., Methods: Between 2008 and 2011, thirty cystine stone patients were involved in our study, and an equal number of age- and gender pair-matched patients with calcium oxalate stones. Non-stone forming individuals were elected as controls. The evaluation included blood chemistry studies and 24-h urine collection in both groups of patients., Results: The cystine stone patients had higher mean values of serum blood urea nitrogen, urate and creatinine levels than patients in other two groups. With respect to urine risk factors, cystine stone patients had higher urinary citrate and lower urinary oxalate and creatinine than calcium oxalate stone patients. When compared to non-stone forming individuals, cystine stone patients had higher urinary urate excretion and lower urinary creatinine excretion. Metabolic abnormalities could be demonstrated in 80 % of the cystine stone patients and in 100 % of the calcium oxalate stone patients. We also compared urine risk factors among cystine stone patients with different urine cystine excretion (<1 mmol/24 h, 1-2 mmol/24 h and >2 mmol/24 h). No significant difference was found in urine risk factors among three groups., Conclusions: This study suggested that cystine stone patients were at greater risk for the loss of renal function than calcium oxalate stone patients, but the risk of the formation of calcium oxalate stones was lower. Our results also indicated that urinary cystine had little or no impact on the excretion of urine chemistries in cystine stone patients.

Introduction: Cystone is an approved Ayurvedic polyherbal proprietary medicine used in India for various urinary disorders, including urolithiasis., Aim: To evaluate the protective effect of Cystone against hyperoxaluria-induced oxidative stress and calcium oxalate crystal deposition in urolithiasis., Methods: Ethylene glycol (EG) (0.75%, V/V) in drinking water was given to rats for 28 days to induce urolithiasis with simultaneous treatment of Cystone (500 and 750 mg/kg body weight), and various urinary risk factors of urolithiasis and antioxidant markers were assessed., Results: EG treatment lead to increased urine volume and lowered urinary pH, along with increased urinary excretion of oxalate, calcium and phosphate in untreated animals. These changes caused extensive calcium oxalate crystal deposition, increased lipid peroxidation and decreased activity of antioxidant enzymes (SOD, catalase and GPx) in the kidney of untreated rats. Cystone prevented these hyperoxaluric manifestations and inhibited calcium oxalate crystal deposition in treated rats at both doses., Conclusions: Cystone therapy provides protection against hyperoxaluria-induced oxidative stress and calcium oxalate crystal deposition by improving renal tissue antioxidant status and diuresis., (Copyright © 2013 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Hyperoxaluria is one of etiologic factors of calcium oxalate kidney stone disease. However, response of renal tubular cells to high-oxalate environment remained largely unknown. We applied a gel-based proteomics approach to characterize changes in cellular proteome of MDCK cells induced by 10mM sodium oxalate. A total of 14 proteins were detected as differentially expressed proteins. The oxalate-induced up-regulation of alpha-enolase in whole cell lysate was confirmed by 2-D Western blot analysis. Interaction network analysis revealed that cellular adaptive response under high-oxalate condition involved stress response, energy production, metabolism and transcriptional regulation. Down-regulation of RhoA, which was predicted to be associated with the identified proteins, was confirmed by immunoblotting. In addition, the up-regulation of alpha-enolase on apical surface of renal tubular epithelial cells was also confirmed by immunoblotting of the isolated apical membranes and immunofluorescence study. Interestingly, blockage of alpha-enolase expressed on the cell surface by antibody neutralization significantly reduced the number of calcium oxalate monohydrate (COM) crystals adhered on the cells. These results strongly suggest that surface alpha-enolase plays an important role as the enhancer of COM crystal binding. The increase of alpha-enolase expressed on the cell surface may aggravate kidney stone formation in patients with hyperoxaluria., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Prevalence of recurrent calcium-oxalate (CaOx) urolithiasis (UL) is up to fivefold higher in Crohn's disease than in the general population. Treatment options are scarce and the risk of recurrent UL or progressive renal CaOx deposition, (oxalosis) based early end-stage renal failure (ESRF), subsequent systemic oxalosis, and recurrence in the kidney graft is pronounced. We aimed to find proof that secondary hyperoxaluria is the main risk factor for the devastating course and correlates with intestinal oxalate absorption., Methods: 24-h urines were collected and analyzed for urinary oxalate (Uox) in 27 pediatric (6-18 years) and 19 adult patients (20-62 years). In the 21 patients (8 adults and 13 children) with hyperoxaluria a [(13)C(2)]oxalate absorption test was performed under standardized dietary conditions., Results: Mean Uox was significantly higher in patients with UL or oxalosis (0.92 ± 0.57) compared with those without (0.53 ± 0.13 mmol/1.73 m(2)/24 h, p<0.05, normal < 0.5). Hyperoxaluria then significantly correlated with intestinal oxalate absorption (p< 0.05)., Conclusion: As UL/oxalosis has major implications for the general health in patients with Crohn's disease (ESRF and systemic oxalosis), new medication, e.g. to reduce intestinal oxalate absorption, is definitely needed.

About 80% of all urological stones are calcium oxalate, mainly caused by idiopathic hyperoxaluria (IH). The increased absorption of oxalate from the intestine is the major factor underlying IH. The continuous self-renewal of the intestinal epithelium is due to the vigorous proliferation and differentiation of intestinal stem cells. If the intestinal stem cell population can acquire the ability to metabolize calcium oxalate by means of oxc and frc transgenes, this will prove a promising new therapy option for IH. In our research, the oxalate-degrading genes of Oxalobacter formigenes (Oxf)-the frc gene and oxc gene-were cloned and transfected into a cultured mouse-derived intestinal SC population to give the latter an oxalate-degrading function. Oxf was isolated and cultivated and the oxalate-degrading genes-frc and oxc-were cloned. The dicistronic eukaryotic expression vector pIRES-oxc-frc was constructed and transferred into the mouse stem cell population. After selection with G418, the expression of the genes was identified. The oxalate-degrading function of transfected cells was determined by transfection into the intestinal stem cell population of the mouse. The change in oxalate concentration was determined with an ion chromatograph. The recombinant plasmid containing oxc and frc genes was transfected into the stem cell population of the mouse and the expression of the genes found normal. The cell population had acquired an oxalate-degrading function. The oxc and frc genes could be transfected into the intestinal stem cell population of the mouse and the cells acquired an oxalate-degrading function.

Background and Objectives: Enteric overabsorption of oxalate may lead to hyperoxaluria and subsequent acute oxalate nephritis (AON). AON related to chronic pancreatitis is a rare and poorly described condition precluding early recognition and treatment., Design, Setting, Participants, & Measurements: We collected the clinical characteristics, treatment, and renal outcome of 12 patients with chronic pancreatitis-associated AON followed in four French renal units., Results: Before AON, mild to moderate chronic kidney disease was present in all patients, diabetes mellitus in eight (insulin [n = 6]; oral antidiabetic drugs [n = 2]), and known chronic pancreatitis in only eight. At presentation, pancreas imaging showed gland atrophy/heterogeneity, Wirsung duct dilation, calcification, or pseudocyst. Renal findings consisted of rapidly progressive renal failure with tubulointerstitial profile. Acute modification of glomerular filtration preceded the AON (i.e., diarrhea and diuretics). Increase in urinary oxalate excretion was found in all tested patients and hypocalcemia in nine (<1.5 mmol/L in four patients). Renal biopsy showed diffuse crystal deposits, highly suggestive of oxalate crystals, with tubular necrosis and interstitial inflammatory cell infiltrates. Treatment consisted of pancreatic enzyme supplementation, oral calcium intake, and an oxalate-free diet in all patients and renal replacement therapy in five patients. After a median follow-up of 7 months, three of 12 patients reached end-stage renal disease., Conclusion: AON is an under-recognized severe crystal-induced renal disease with features of tubulointerstitial nephritis that may occur in patients with a long history of chronic pancreatitis or reveal the pancreatic disease. Extrinsic triggering factors should be prevented.

Background: Renal calcium oxalate (CaOx) crystal deposition is associated with epithelial injury and movement of inflammatory cells into the interstitium. We have proposed that oxalate (Ox)- and CaOx crystal-induced injury is most likely caused by reactive oxygen species (ROS) produced by activation of membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase., Methods: Present study was undertaken to determine the effect of NADPH oxidase inhibitor apocynin on the expression of kidney injury molecule-1 (KIM-1) and renal CaOx crystal deposition in rats with hyperoxaluria. We also investigated the urinary excretion of KIM-1, osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1) and renal expression of OPN and ED-1. Male Sprague-Dawley rats were fed a diet containing 5% hydroxyl-L-proline (HLP) and 4 mmol apocynin to drink for 28 days. Urine was collected on Days 7, 14, 21 and 28. After that, rats were sacrificed and their kidneys processed for various microscopic and molecular investigations., Results: HLP consumption produced heavy deposits of CaOx crystals. Renal expression of KIM-1 and OPN and urinary excretion of KIM-1, OPN, H(2)O(2) and MCP-1 was significantly increased. ED-1-positive cells migrated into renal interstitium. Apocynin treatment caused significant reduction of crystal deposits, injured and dilated tubules; renal expression of KIM-1, OPN and ED-1 and urinary excretion of KIM-1, OPN, MCP-1 and H(2)O(2). Apocynin had no effect on the urinary excretion of Ox., Conclusions: This is the first study of urinary excretion and renal expression of KIM-1 in association with renal CaOx crystal deposition, experimental or clinical. The results indicate that NADPH oxidase inhibition leads to reduction in KIM-1 expression and urinary excretion as well as renal CaOx crystal deposition. KIM-1 is an important marker of renal epithelial injury. The results provide further support to our proposal that renal epithelial injury is critical for crystal retention and that injury is in part caused by the production of ROS with the involvement of NADPH oxidase.

Objectives: Subepithelial hydroxyapatite calcification of renal papilla is thought to be involved in the formation of calcium oxalate monohydrate (COM) papillary calculi. To assess the mechanism of formation, we sought to correlate the fine structure of papillary renal calculi with specific pathophysiologic conditions and urinary alterations., Methods: The study included 831 COM papillary renal calculi with established fine inner structures. A total of 24 patients with chronic stone formation were randomly selected, and their urine was collected and analyzed. The case history and lifestyle habits of these patients were obtained., Results: The 831 papillary calculi could be classified into 1 of 4 main groups. Type I included small calculi in which COM columnar crystals begin to develop in the concave zone in close contact with papillary tissue. Type II calculi contained a hydroxyapatite core located in or near the concave zone. Type III consisted of calculi that developed on the tip of the papillae and in the concave zone, containing hydroxyapatite, calcified tissue, and calcified tubules. Type IV consisted of papillary calculi in which the core, which is situated near, but not in, the concave zone, is formed by intergrown COM crystals and organic matter. Many factors, including urinary alterations (eg, hyperoxaluria), associated diseases (eg, hypertension, diabetes), and consumption or exposure to cytotoxic substances (eg, analgesic abuse) were associated with these types of calculi., Conclusions: Our findings have indicated that injury is the first cause of papillary COM calculus formation, with the location of the injury determining the morphology of the resulting calculus., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: 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., Methods: 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)., Results: 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., Conclusions: 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., (© 2010 The Japanese Urological Association.)

Objectives: To test the hypothesis that patients with hyperoxaluria, who modified their dietary calcium intake, would reduce their urinary oxalate excretion without raising their urinary calcium excretion. Diet is a major factor in idiopathic calcium oxalate urolithiasis, yet controversy exists regarding the ideal clinical recommendations. Approximately 20% of patients with calcium oxalate stone formation have hyperoxaluria (> or = 45 mg oxalate/d). Calcium supplements to bind dietary oxalate have been suggested, but clinical evidence of this therapy is lacking., Methods: Of 144 adult patients with stone formation seen by a registered dietitian from September 2006 to September 2007, 26 (18%) had hyperoxaluria on > or = 1 24-hour urinalyses. Of those with > or = 2 complete 24-hour collections and whose hyperoxaluria was observed before their last visit with the registered dietitian, 22 patients were identified. The patients were retrospectively separated into 2 groups according to whether they had been advised dietary changes alone (diet group, n = 10) or calcium citrate with meals, in addition to the dietary changes (supplement group, n = 12). The mean follow-up time was 317 and 266 days for the diet and supplement groups, respectively. Statistical comparisons within and between groups were made for urinary risk factors., Results: Urinary oxalate excretion decreased from 56 to 43 mg/d and from 60 to 46 mg/d in the diet and supplement groups, respectively (P = .003 and P = .038, respectively). Calcium oxalate supersaturation decreased from 3.48 to 1.83 and from 2.37 to 1.52 in the diet and supplement groups, respectively (P = .043 and P = .002, respectively). Urinary calcium excretion did not change in either group., Conclusions: Gastrointestinal binding of oxalate by calcium is an effective clinical strategy for hyperoxaluria, whether mediated by calcium citrate with meals or by inclusion of calcium-containing foods with meals.

Vitamin E was previously reported to reduce calcium oxalate (CaOx) crystal formation. This study explored whether vitamin E deficiency affects intrarenal oxidative stress and accelerates crystal deposition in hyperoxaluria. The control (C) group of rats received a standard diet and drinking water, while the experimental groups received 0.75% ethylene glycol (EG) in drinking water for 42 days. Of the latter, one group received a standard diet (EG group), one received a low-vitamin E (LE) diet (EG+LE group), and the last received an LE diet with vitamin E supplement (4 mg) (EG+LE+E group). The C+LE and C+LE+E groups were the specific controls for the last two experimental groups, respectively. In a separate experiment, EG and EG+LE rats were studied on days 3-42 to examine the temporal relationship between oxidative change and crystal formation. Urinary biochemistry and activity/levels of antioxidative and oxidative enzymes in glomeruli and tubulointerstitial specimens (TIS) were examined. In EG rats, CaOx crystal accumulation was associated with low antioxidative enzyme activity in TIS and with increased oxidative enzyme expression in glomeruli. In the EG+LE group, marked changes in antioxidative and oxidative enzyme levels were seen and correlated with massive CaOx deposition and tubular damage. The increased oxidative stress seen with EG+LE treatment was largely reversed by vitamin E supplementation. A temporal study showed that decrease in antioxidative defense and increased free radical formation in the EG+LE group occurred before crystal deposition. This study shows that low vitamin E disrupts the redox balance and causes cell death, thereby favoring crystal formation.

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.

Oxalate, one of the major constituents of renal stones is known to induce free radicals which damage the renal membrane. Damaged epithelia might act as nidi for stone formation aggravating calcium oxalate precipitation during hyperoxaluria. In the present study, the beneficial effects of fucoidan on oxalate-induced free radical injury were investigated. Male Wistar rats were divided into four groups. Hyperoxaluria was induced in two groups by administration of 0.75% ethylene glycol in drinking water for 28 days and one of them was treated with fucoidan from Fucus vesiculosus at a dose of 5 mg/kg b.wt subcutaneously commencing from the 8th day of induction. A control and drug control (fucoidan alone) was also included in the study. The extent of renal injury in hyperoxaluria was evident from the increased activities of alkaline phosphatase, gamma-glutamyl transferase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase in urine. There was a positive correlation between plasma malondialdehyde levels and renal membrane damage indicating a striking relation between free radical formation and cellular injury. Increased protein carbonyl and decreased thiols further exemplified the oxidative milieu prevailing during hyperoxaluria. Decreased renal membrane ATPases accentuated the renal membrane damage induced by oxalate. Renal microscopic analysis showed abnormal findings in histology as an evidence of oxalate damage. The above biochemical and histopathological discrepancies were abrogated with fucoidan administration, indicating its protective role in oxalate mediated peroxidative injury.

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.

Environment and diet have a major role in calcium nephrolithiasis by affecting urine saturation, but this is not enough to cause lithogenesis; the crystals must adhere to the tubular epithelium (TE), but it is hard to say how environment and nutrition may be involved in this step. The hypothesis that TE damage (known to enhance crystal attachment) is lithogenic in mild hyperoxaluria was tested. Mild hyperoxaluria was induced in male Wistar rats using ethylene glycol (EG; 0.5% in water) for 21 d, and TE damage was induced by intraperitoneal administration of hexachloro-1:3-butadiene (HCBD; an industrial nephrotoxin) at 10, 25, and 50 mg/kg body wt on days 7 and 14. These EG and HCBD concentrations were chosen to span from suboptimal to very low doses as far as effects on crystalluria and TE damage are concerned. Enzymuria, proteinuria, oxaluria, crystalluria, and renal pathology were investigated. All HCBD dosages induced crystalluria in mildly hyperoxaluric rats, but no intrarenal crystals were found. EG alone induced very mild hyperoxaluria but no damage to the renal tubule observable on transmission electron microscopy, and it did not cause crystalluria or intrarenal crystals. HCBD with the concomitant administration of EG caused apoptosis of the TE at the two highest dosages after the second injection. Apoptosis did not correlate with crystalluria. A TE toxin is needed for crystallogenesis to occur in borderline metabolic conditions. It may take more than just a metabolic predisposition for calcium nephrolithiasis to occur, and the second hit could come from an environmental pollutant such as HCBD.

Background: Increased rates of either oxalate absorption or endogenous oxalate synthesis can contribute to hyperoxaluria, a primary risk factor for the formation of calcium oxalate-containing kidney stones. This study involves a comparative assessment of oxalate absorption and endogenous oxalate synthesis in subpopulations of stone formers (SFs) and non-stone formers (NSFs) and an assessment of the effect of ascorbate supplementation on oxalate absorption and endogenous oxalate synthesis., Methods: Twenty-nine individuals with a history of calcium oxalate kidney stones (19 men, 10 women) and 19 age-matched NSFs (8 men, 11 women) participated in two 6-day controlled feeding experimental periods: ascorbate-supplement (2 g/d) and no-supplement treatments. An oxalate load consisting of 118 mg of unlabeled oxalate and 18 mg of 13C2 -oxalic acid was administered the morning of day 6 of each experimental period., Results: Mean 13C2 -oxalic acid absorption averaged across the ascorbate and no-supplement treatments was significantly greater in SFs (9.9%) than NSFs (8.0%). SFs also had significantly greater 24-hour post-oxalate load urinary total oxalate and endogenous oxalate levels with both treatments. Twenty-four-hour urinary total oxalate level correlated strongly with both 13C2 -oxalic acid absorption (SFs, r = 0.76; P < 0.01; NSFs, r = 0.62; P < 0.01) and endogenous oxalate synthesis (SFs, r = 0.95; P < 0.01; NSFs, r = 0.92; P < 0.01)., Conclusion: SFs are characterized by greater rates of both oxalate absorption and endogenous oxalate synthesis, and both these factors contribute to the hyperoxaluric state. The finding that ascorbate supplementation increased urinary total and endogenous oxalate levels suggested that this practice is a risk factor for individuals predisposed to kidney stones.

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.

Background: Hyperoxaluria is a major predisposing factor in calcium oxalate urolithiasis. The aim of the present study was to clarify the role of dietary oxalate in urinary oxalate excretion and to assess dietary risk factors for hyperoxaluria in calcium oxalate stone patients., Methods: Dietary intakes of 186 calcium oxalate stone formers, 93 with hyperoxaluria (>or=0.5 mmol/day) and 93 with normal oxalate excretion (<0.4 mmol/day), were assessed by a 24-hour weighed dietary record. Each subject collected 24-hour urine during the completion of the food record. Oxalate content of foods was measured by a recently developed analytical method., Results: The mean daily intakes of energy, total protein, fat and carbohydrates were similar in both groups. The diets of the patients with hyperoxaluria were estimated to contain 130 mg/day oxalate and 812 mg/day calcium as compared to 101 mg/day oxalate and 845 mg/day calcium among patients without hyperoxaluria. These differences were not significant. The mean daily intakes of water (in food and beverages), magnesium, potassium, dietary fiber and ascorbic acid were greater in patients with hyperoxaluria than in stone formers with normal oxalate excretion. Multiple logistic regression analysis revealed that urinary oxalate excretion was significantly associated with dietary ascorbate and fluid intake, and inversely related to calcium intake. Differences of estimated diet composition of both groups corresponded to differences in urinary parameters., Conclusions: These findings suggest that hyperoxaluria predominantly results from increased endogenous production and from intestinal hyperabsorption of oxalate, partly caused by an insufficient supply or low availability of calcium for complexation with oxalate in the intestinal lumen.

Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaOx) being the predominant constituent of renal stones. Current in vitro evidence implicates osteopontin (OPN) as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention. To determine the importance of OPN in vivo, hyperoxaluria was induced in mice targeted for the deletion of the OPN gene together with wild-type control mice. Both groups were given 1% ethylene glycol, an oxalate precursor, in their drinking water for up to 4 wk. At 4 wk, OPN-deficient mice demonstrated significant intratubular deposits of CaOx crystals, whereas wild-type mice were completely unaffected. Retained crystals in tissue sections were positively identified as CaOx monohydrate by both polarized optical microscopy and x-ray powder diffraction analysis. Furthermore, hyperoxaluria in the OPN wild-type mice was associated with a significant 2- to 4-fold upregulation of renal OPN expression by immunocytochemistry, lending further support to a renoprotective role for OPN. These data indicate that OPN plays a critical renoprotective role in vivo as an inhibitor of CaOx crystal formation and retention in renal tubules.

Urolithiasis is one of the third most common afflictions found in humans. The efficacy of the two Siddha drugs, Aerva lanata and Vediuppu chunnam as antilithic agents using a urolithic rat model were tested in this study. Hyperoxaluria was induced in rats using 0.75% ethylene glycol in drinking water. Aerva lanata(3.0 mg kg(-1)body weight) and Vediuppu chunnam (3.5 mg kg(-1)body weight) were given orally for 28 days. Urinary risk factors of urolithiasis were monitored at the end of 7th, 14th, 21st and 28th days. Urinary volume was increased in hyperoxaluric as well as drug-treated rats. Increased urinary excretion of calcium, oxalate, uric acid, phosphorus and protein in hyperoxaluric rats was brought down significantly by the administration of A. lanata or Vediuppu chunnam. Decreased magnesium excretion in hyperoxaluric rats was normalized by drug treatment. The drug increases the urine volume, thereby reducing the solubility product with respect to calcium oxalate and other crystallizing salts such as uric acid, which may induce epitaxial deposition of calcium oxalate. Drug alone treated rats did not show any adverse effects. Combination therapy was found to be more effective and this indigenous medicine can be used successfully as an antilithic agent., (Copyright 2001 Academic Press.)

Objective: 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., Results: 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., Conclusion: 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.

Background: A patient with cystic fibrosis (CF) and repeated calcium oxalate renal stones prompted us to investigate other children for risk factors for this recognised complication of CF., Methods: Twenty four hour urinary excretion of calcium, oxalate, and glycolate was measured in children with CF and no symptoms of renal tract stones. Normal diet and treatments were continued., Results: In 26 children (aged 5-15.9 years) oxalate excretion was correlated with age; 14 of 26 children had oxalate excretion above an age appropriate normal range. There was a positive correlation between oxalate excretion and glycolate excretion. Mean calcium excretion was 0.06 mmol/kg/24 h with 21 of 24 children having calcium excretion below the normal range., Conclusions: Hyperoxaluria may reflect malabsorption although correlation between excretion of oxalate and glycolate suggests a portion of the excess oxalate is derived from metabolic processes. The hypocalciuria observed here may protect children with CF from renal stones.

Objective: To determine the metabolic characteristics of elderly patients with recurrent calcium oxalate stones., Patients and Methods: Metabolic abnormalities were investigated in 88 patients with recurrent calcium oxalate stones, including 70 aged <60 years and 18 aged >/=60 years. The frequency of each metabolic abnormality and the value of each urinary constituent were compared among subgroups of age and gender., Results: Hyperoxaluria was the most common abnormality, present in 56% and 67% of patients aged <60 and >/=60 years, respectively. Hyperuricosuria was significantly more common in older than in younger patients. There were no significant differences in the frequencies of hypercalciuria and hypocitraturia between the age groups. The urinary excretion of oxalate and the ratio of oxalate to creatinine were significantly greater in older than in younger men. The frequency of low urine volume was lower in older than in younger patients and the mean urinary volume was also greater in the older group., Conclusions: Hyperuricosuria and hyperoxaluria seem to be essential risk factors for calcium oxalate stone formation in elderly patients. Urinary oxalate excretion is significantly greater in older than in younger stone formers and is more prominent in men.

Aim: The gamma-carboxy glutamic acid (gla) containing proteins have been shown recently to have a role in calcium oxalate urolithiasis. Therefore, the effect of carboxylation on calcium oxalate monohydrate (COM) binding was studied in experimental hyperoxaluria., Methods: Urolithiasis was induced in experimental rats by oral feeding 1% ethylene glycol (EG). The microsomes were carboxylated in presence of reduced vitamin K and COM binding activity was determined., Results: Microsomal proteins adsorbed maximally with COM crystals. On carboxylation of the renal microsomal proteins, a significant increase in the COM crystal adsorption by 2.5-fold (p < 0.001) was observed in the hyperoxaluric condition. Further cyboxylated microsomes of EG-treated rats showed significant binding with calcium oxalate., Conclusion: This study presents an increased COM adsorption after the carboxylation of renal microsomes of hyperoxaluric rats.

We studied 476 patients with idiopathic renal calcium stone disease (286 M, 190 F) while they ate their customary diets. Each subject collected a 24-hour urine sample and completed a dietary diary for a 3-day period. Daily urinary oxalate excretion (M 0.24 +/- 0.15 mg/dl, F 0.23 +/- 0.15 mg/dl) and nutrient intake values were calculated and multiple regression analyses were performed. Daily urinary oxalate excretion was significantly (p < 0.001) related to urinary volume (R = 0.24), vitamin C intake (R = 0.33) and body mass index (R = 0.37) and inversely related to calcium intake (R = -0.35). We conclude that urinary oxalate reflects endogenous oxalate production, presumably related to body size, but also intestinal absorption of oxalate, related to dietary intake and to the effect of dietary calcium intake which reduces intestinal oxalate absorption.

Calcium oxalate (CaOx) nephrolithiasis was induced in male Sprague-Dawley rats by administration of 0.75% ethylene glycol. Urinary excretion of lactate dehydrogenase (LDH) was used as a marker of cellular injury. Lipid peroxides (LP), as marker for free radical injury, were measured as malondialdehyde (MDA) in urine and the kidneys. Urinary oxalate (Ox), LDH, LP, CaOx crystals, and renal LP and CaOx crystal deposits were examined on day 0, 5, 30 and 60 of the experiment. There were significant differences between control and experimental rats in all the parameters except LDH which did not show a significant increase after 15 days. Subconfluent cultures of MDCK and LLCPK1 cells were exposed to various concentrations of oxalate and/or 500 fg/ml CaOx crystals. Cell viability was assayed by trypan blue exclusion, cellular injury was determined by measuring LDH in the media, and free radical injury was measured as MDA contents of the cells. On exposure to both Ox and/or CaOx crystals trypan blue exclusion decreased and LDH and MDA increased significantly in both tissue cultures. LLC-PK1 appeared more sensitive. The results indicate that both oxalate and calcium oxalate crystals are injurious to renal epithelial cells in the kidneys as well as in culture.

Purpose: To determine if lipid peroxidation plays a role in renal injury associated with experimental nephrolithiasis., Materials and Methods: Hyperoxaluria was produced in rats by ethylene glycol in drinking water. At 15, 30 and 60 days of treatment, urinary oxalate, lipid peroxide, calcium oxalate crystals, enzymes and tissue lipid peroxide were measured., Results: Urinary oxalate increased significantly at all time periods and was associated with crystalluria. Lipid peroxides in kidney tissue and urine increased at all time periods. Tissue calcium oxalate crystal deposits from 0 to 1+ were present on day 15, but present in all animals on days 30 and 60. Renal tubular cell damage was confirmed by an increase in urinary marker enzymes., Conclusions: Renal cell damage is associated with lipid peroxide production indicating cell injury due to the production of free radicals. The damage appears due primarily to hyperoxaluria and is augmented by crystal deposition in the renal tubules.

The in vivo effect of cyclosporin A (CsA) on renal calcium oxalate (CaOx) crystal retention in experimental hyperoxaluric rats was investigated. Further, the effect of pretreatment of vitamin E on the above conditions was also studied. Male Wistar rats were divided into two major groups each containing 40 rats. One of the groups was pretreated with vitamin E. Both major groups were then subgrouped into four groups: group 1 received the vehicle (olive oil); group 2 received CsA in olive oil (50 mg/kg); group 3 received 3% ammonium oxalate (AmOx), and group 4 received CsA + AmOx. Nephrotoxicity was assessed by the activities of urinary marker enzymes and also by histopathology. Urinary oxalate excretion as well as the activities of lactate dehydrogenase, gamma-glutamyltranspeptidase, alkaline phosphatase and inorganic pyrophosphatase enzymes were elevated either in CsA-alone or AmOx-alone treated groups. On combined administration of both CsA and AmOx, further elevations of these enzymes were observed. Urinary excretion of oxalate concentration positively correlated with urinary excretion of these enzymes. Deposition of CaOx crystals was seen only in the kidneys of rats that received combined treatment. On pretreatment with vitamin E the observed increased urinary activities of the enzymes and oxalate, histopathological changes and the deposition of CaOx crystals by administration of CsA in hyperoxaluria were prevented suggesting that vitamin E could be supplemented to prevent CsA-induced membrane damage.

Oxalosis, or calcium oxalate deposition in the tissues, may develop in patients with inherited disorders of oxalate metabolism or can occur secondary to other diseases. In this study, a case of renal oxalosis probably secondary to excessive parenteral vitamin C administration in a patient with acute post-traumatic oliguric renal failure is reported. Oxalate deposits may have contributed to further worsening and delayed recovery of renal function. The elimination of the source of excess vitamin C and its presumed effect on oxalate production, together with enhanced removal of oxalate during aggressive dialysis, resulted in prompt recovery of renal function. Secondary oxalosis represents a possible cause of delayed recovery of renal function in patients with acute renal failure who are receiving vitamin C supplementation if excess dosage of that supplementation is given. Vitamin C supplementation, if utilized, should be carefully monitored in patients receiving artificial renal replacement therapy.

Oxalosis is an unusual metabolic disease that results either from an inherited hepatic enzyme deficiency or as the result of poor oxalate clearance during chronic hemodialysis. We present two cases of oxalosis and describe the hand manifestations of this condition and their treatment. One patient had painful, progressive gangrene, whereas in the other the disease took an indolent course with small palmar crystalline deposits.