Search

Objectives: To clarify the role of renal tubular cell (RTC) injury and oxidative stress in the early stage of renal calcium oxalate crystal formation in a mouse model. Methods: Daily intra-abdominal injections of glyoxylate (1.35 mmol/kg/day) into 8-week-old mice were carried out over 6 days. Kidneys were extracted before and at 6, 12 and 24 h and 3 and 6 days after glyoxylate injection. Crystal formation was detected using Pizzolato staining and polarized light optical microscopy. Immunohistochemical staining and western blotting of superoxide dismutase, and 4-hydroxynonenal and malondialdehyde were carried out in order to observe oxidative stress and lipid peroxidation, respectively. RTC microstructural damage and crystal nuclei formation were observed using transmission electron microscopy. To ameliorate RTC injury, mice were treated with green tea 1 week before and 1 week after glyoxylate administration. The number of crystals and RTC damage were observed and comparisons were made between glyoxylate-treated mice with and without green tea administration. Results: Oxidative stress and lipid peroxidation were observed after 6 h. Crystal nuclei containing collapsed mitochondria and fallen microvilli appeared in the renal distal tubular lumen after 24 h. Crystals occupying the tubular lumen were detected on day 3. The number of crystals in mice receiving green tea was significantly lower than in those receiving glyoxylate alone. Conclusions: RTC injury, especially mitochondrial damage, and oxidative stress induce the early stage of calcium oxalate crystal formation in mice.

We previously established a mouse kidney stone formation model and showed that mice have a higher tolerance to stone formation than rats. Furthermore, we showed that the generated calcium oxalate crystal deposits could be eliminated after several days. This study investigated the transcriptome of stone formation and elimination in the mouse kidney based on gene selection using a microarray technique. Eight-week-old male C57BL/6N mice were administered 80 mg/kg glyoxylate for 15 days, and kidney calcium oxalate crystal depositions had increased by day 6; thereafter, depositions decreased gradually and had almost disappeared by day 15. On microarray analysis, mRNA expression in the crystal-formed kidneys showed the significant expression of 18,064 genes. Thirty-one, 21, and 25 genes showed at least a 2-fold increased expression during the experimental course (days 3-15), stone formation phase-specific (days 3-6), and stone elimination phase-specific (days 9-15) stages, respectively. Among these genes, those related to chemotaxis and monocyte/macrophage activation were identified. Gene ontology analysis to identify overexpressed genes highlighted categories related to inflammation, immune reactions and the complement activation pathway. Quantitative PCR of 17 previously reported stone-related genes with a significant expression on microarray analysis showed significantly increased chemokines, stone matrix proteins, and their receptors; the significant decrease of several types of transporters and superoxide dismutase; and the persistently high expression of Tamm-Horsfall protein throughout the experiment. In conclusion, inflammation and immune reactivity through macrophage migration are involved in stone formation and elimination in mouse kidneys.

Objective: Osteoporosis is associated with the pathogenesis and risk of urolithiasis, which is higher among postmenopausal women (as opposed to premenopausal). Bisphosphonates potently inhibit bone resorption, and are used in the management of bone disease. We investigated the ability of a bisphosphonate to prevent calcium stone formation. Methods: We studied 12 postmenopausal women (63.8 ± 7.3 years) who were not receiving osteoporosis therapy, and had stones comprised of calcium phosphate (CaP; n = 3), calcium oxalate (CaOx; n = 3) and CaP + CaOx (n = 6). We measured bone mineral density (BMD), serum and urinary values in 24-hour urine specimens before and 3 months after the oral administration of 5 mg/day of alendronate (ALN). The indexes of the ionic activity product of calcium oxalate, AP(CaOx), and of calcium phosphate, AP(CaP), were estimated using the Tiselius method. Results: ALN significantly reduced the AP(CaP) index (1.53 ± 1.37 to 0.89 ± 0.81, p Conclusion: The results suggested that ALN not only improves BMD and osteoporosis, but also reduces the risk of calcium phosphate stone formation in postmenopausal women.

Objectives: The transcription factor nuclear factor–κB (NF–κB) is involved in inflammatory and immune responses through the induction of various cytokines and growth factors. Recently, the coordinated action of NF–κB and activator protein-1 was reported in osteopontin (OPN) expression. In the present study, we demonstrated that oxalate induces OPN expression by activating NF–κB in renal tubular cells. Furthermore, we investigated the inhibitory effect of N-acetyl-L-cysteine (NAC) on NF–κB activation in the human renal tubular cell line. Methods: All of the experiments were carried out using human kidney-2 cells, which are human proximal tubular epithelial cells immortalized by transduction with the human papillomavirus 16E6/E7 gene. The time-dependent extraction of total protein was performed after the uptake of 0.5 mM oxalate by the cells. The NF–κB activation and OPN expression were examined by western blotting and immunocytochemistry. Results: As a result of oxalate stimulation, the amount of p65 subunit in the nucleus increased significantly (P

Objectives: To clarify the influence of long-term bed rest on renal stone formation and to analyze the mechanism of bed-rest-induced stone formation and prevention by bisphosphonate and bed-rest exercise. Methods: Twenty-five men aged 26–48 years and divided into control (CON: n = 9), exercise (EX: n = 9), and pamidronate (PMD: n = 7) groups, rested on a 6° head-down tilt bed for 90 days. The exercise group carried out resistive exercise every 3 days. Pamidronate (60 mg) was intravenously given 2 weeks before the initiation of bed rest. Abdominal X-ray examination and urine biochemistry were carried out during 90 days of bed rest and 90 days of reloading. Results: Renal stone formation was observed in two (22.2%) and four (44.4%) subjects in the control and exercise groups, respectively. No stone was seen in the pamidronate group. In the exercise group, urinary oxalate and phosphate excretion were significantly higher than in the control group. In the pamidronate group, urinary calcium excretion and relative supersaturation of calcium oxalate and brushite were lower than in the control group throughout the bed-rest and recovery period. Conclusion: Long-term bed-rest-induced renal stone formation was found to be induced by increased urinary calcium and subsequent crystal formation of calcium oxalate and calcium phosphate. Exercise during bed rest for the prevention of bone mineral loss and contracture might increase the risk of renal stone formation. Pamidronate is useful for the prevention of renal stone formation during and after bed rest.

An important process in kidney stone formation is the conversion of retentive crystals in renal tubules to concrete stones. Osteopontin (OPN) is the major component of the kidney calcium-containing stone matrix. In this study, we estimated OPN function in early morphological changes of calcium oxalate crystals using OPN knockout mice: 100 mg/kg glyoxylate was intra-abdominally injected into wildtype mice (WT) and OPN knockout mice (KO) for a week, and 24-h urine oxalate excretion showed no significant difference between WT and KO. Kidney crystal depositions were clearly detected by Pizzolato staining but not by von Kossa staining in both genotypes, and the number of crystals in KO was significantly fewer than in WT. Morphological observation by polarized light optical microphotography and scanning electron microphotography (SEM) showed large flower-shaped crystals growing in renal tubules in WT and small and uniform crystals in KO. X-ray diffraction detected the crystal components as calcium oxalate monohydrate in both genotypes. Immunohistochemical staining of OPN showed that the WT crystals contained OPN protein but not KO crystals. We concluded that OPN plays a crucial role in the morphological conversion of calcium oxalate crystals to stones in mouse kidneys.

the predominant component (>60%) of their stone. Patients with

The incidence of urolithiasis has increased in the industrialized nations. However, both the pathogenesis and methods for its prevention remain to be clarified. We demonstrate that the antioxidative effect of green tea decreases the formation of calcium oxalate stones, OPN (osteopontin) expression, and apoptosis, and increases SOD (superoxide dismutase) activity in rat kidney tissues. The inhibitory effect of green tea on calcium oxalate urolithiasis is most likely due to its antioxidative effects. Therefore, we examined oxidative stress in vivo applied to Madin-Darby canine kidney (MDCK) cells, to which catechin, an antioxidant, was added. To evaluate the effects of oxidative stress on MDCK cells, we use a hypoxic condition because hypoxia is known to lead to oxidative stress. Confluent cultures of MDCK cells were exposed to (-)epigallocatechin 3 gallate (EGCG) (0, 0.1, 0.5, 5.0 mg/ml) for 2, 4, 8 or 16 h to determine changes in protein secretion and apoptosis. OPN protein expression was observed in MDCK cells of all 16 groups. The levels of expression of OPN protein were the same among all groups. In all groups, SOD protein expression was observed. In the groups exposed to EGCG 0.5, 5.0 mg/ml, SOD staining was more enhanced than in the EGCG 0 and 0.1 mg/ml groups. No deposits were detected in any of the 16 groups. RT-PCR was performed to detect sequences from OPN (979 bp) and SOD (447 bp). Quantitative analyses showed that SOD activity decreased gradually in all groups. Only in the EGCG 0 mg/ml 16 h group were TUNEL-positive cells observed. In the other groups, TUNEL-positive cells were not detected. EGCG used as an antioxidant protects renal tubular cell from cellular injury caused by oxidative stress through SOD protein expression.

Background: At present, immunotherapeutic agents such as bacillus Calmette-Guerin (BCG) and anti-tumor chemotherapeutic agents in saline are used intravesically in patients with bladder carci- noma. However, of greater significance is the possibility that the saline vehicle may itself promote carcinoma development in the bladder. Methods: The potential promoting effects of intravesical instillation of saline were assessed in female F344 rats. The animals were divided into 3 groups, all of which received 0.05% N-butyl-N- (4-hydroxybutyl)nitrosamine (BBN) in their drinking water for the first 10 weeks. They were then maintained without further treatment (group 1) or received intravesical instillations of 0.3 mL of saline or distilled water once a week for 6 weeks, 15 weeks after the end of the BBN treatment (groups 2 and 3). At 32 weeks, all the animals were killed and examined immunohistochemically with proliferating cell nuclear antigen (PCNA) antibody, as well as by routine histopathologic examination. Results: Both the incidence and the number of bladder carcinomas were higher in the animals that received instillations of saline than in those who did not receive the instillations. Significant increases in tumor size were also noted for the saline-treated groups, although this was not linked with the PCNA labeling index. Conclusions: The results indicate that saline is a promoter of urinary bladder carcinogenesis either because of the catheterization or the fluid itself.

Background Residents (n = 157) of Kamisu City, Ibaraki, Japan, were orally exposed to diphenylarsinic acid (DPAA) via the ingestion of contaminated underground water. Subsequently, a clinical syndrome associated with a variety of cerebellar and brainstem symptoms, was observed in 20 of the 30 residents who consumed high concentrations of DPAA in the contaminated well water. While the clinical symptoms of DPAA were defined, the toxicokinetics of DPAA remained unclear. Methods In order to investigate the underlying toxicokinetics of DPAA, we collected serum and cerebrospinal fluid (CSF) samples from 5 patients with DPAA intoxication, and attempted to estimate the half-life of serum DPAA and the CSF/serum ratio of DPAA. Results DPAA, and its derivatives, such as phenylmethylarsinic acid (PMAA) and phenylarsinic acid (PAA), were detected in serum from residents exposed to DPAA. Serum DPAA was observed for > 200 days after the last ingestion of contaminated water. The half-life of serum DPAA was 22.5 days in children and 39.4 days in youths and adults, which was nearly double that observed in children. DPAA was found in CSF, and the CSF/serum ratios of DPAA in 2 patients were 3.0% and 3.7%, respectively, suggesting that this toxicant is able to cross the blood–brain barrier. Conclusion An established animal model of DPAA intoxication was examined regarding the toxicokinetics, distribution and direct DPAA accumulation in the cerebrum. On the basis of existing animal data, and the present results arising from human subjects, the development of new therapies for DPAA intoxication should be enhanced, such as accelerated DPAA excretion.

Clinical managements of chronic pain in rheumatoid arthritis (RA) and osteoarthritis (OA) require deep understanding of patients. Since medication is mostly set under the guidelines of RA and OA to control of pain, but personal characteristics is not so much paid attention in terms of preferences. In the present study, alcohol preference was analyzed in the patients prescribed pregabalin and tramadol/acetaminophen in our hospital. All consecutive patients were requested to write up the medical questionnaire sheet before medical examination. The responses to this sheet are completely subjective. The close relation between habitual alcohol drinking and adherence of analgesics was observed in terms of adherence of pregabalin and tramadol/acetaminophen. That is, in OA patients, alcohol drinkers showed good adherence to pregabalin and also tramadol/acetaminophen. However, in RA patients, alcohol drinkers showed higher ratio to good adherence, but there was no significance between alcohol drinkers and non-alcohol drinkers. When total patients were analyzed, alcohol drinkers showed significantly higher adherence with pregabalin and also tramadol/acetaminophen. Therefore, it is recommended that precise personal preference had better to be taken in medical examination before prescription of pregabalin and tramadol/ acetaminophen in RA and OA patients.

Renal tubular cell injury induced by oxalate plays an important role in kidney stone formation. Water containing oxygen nano-bubbles (nanometer-sized bubbles generated from oxygen micro-bubbles; ONB) has anti-inflammatory effects. Therefore, we investigated the inhibitory effects of ONB water on kidney stone formation in ethylene glycol (EG)-treated rats. We divided 60 rats, aged 4 weeks, into 5 groups: control, the water-fed group; 100 % ONB, the 100 % ONB water-fed group; EG, the EG treated water-fed group; EG + 50 % ONB and EG + 100 % ONB, water containing EG and 50 % or 100 % ONB, respectively. Renal calcium oxalate (CaOx) deposition, urinary excretion of N-acetyl-β-d-glucosaminidase (NAG), and renal expression of inflammation-related proteins, oxidative stress biomarkers, and the crystal-binding molecule hyaluronic acid were compared among the 5 groups. In the control and 100 % ONB groups, no renal CaOx deposits were detected. In the EG + 50 % ONB and EG + 100 % ONB groups, ONB water significantly decreased renal CaOx deposits, urinary NAG excretion, and renal monocyte chemoattractant protein-1, osteopontin, and hyaluronic acid expression and increased renal superoxide dismutase-1 expression compared with the EG group. ONB water substantially affected kidney stone formation in the rat kidney by reducing renal tubular cell injury. ONB water is a potential prophylactic agent for kidney stones.

treated mice had stones. The number of stones ranged from 16-45 (25.86 / 10.96, n 181). All stones were in the small group, except one 3mm stone. Mean stone weight was 29.2 / 14.8mg (range 14.2-52.3mg). Mean bladder weight in mice with stones was 65.4 / 18.6mg (range 42.2-101.1) and without stones it was 18.6 / 5.2mg (range 10.2-25.2mg). Stone number, stone weight, and bladder weight were significantly different between the two groups (two-tailed t-test, p 0.05) and the difference in stone burden between the groups was clearly evident by micro CT. There was no significant difference in cystine concentration (nmol/mg creatinine) between the two groups (5,410 / 2,124 versus 4,343 / 1,577), suggesting that urine was saturated with cystine in both groups. CDME treatment had no side effects. A dose of 5 mg/ml administered in the water supply also had beneficial effects and no side effects. CONCLUSIONS: CDME-treated mice had a significantly smaller stone mass and stone size, strongly suggesting that CDME is inhibiting, but not preventing, the growth of cystine crystals into stones. While current treatments for cystinuria in humans have poor compliance and multiple side effects, CDME in mice had therapeutic potential and was without adverse effects. Further research is warranted to determine if CDME may provide a new treatment approach for cystinuria, which is clinically invaluable, since cystinuria patients are at risk for multiple surgeries and renal insufficiency.

Renal tubular cell injury induced by oxidative stress via mitochondrial collapse is thought to be the initial process of renal calcium crystallization. Mitochondrial collapse is generally caused by mitochondrial permeability transition pore (mPTP) opening, which can be blocked by cyclosporine A (CsA). Definitive evidence for the involvement of mPTP opening in the initial process of renal calcium crystallization, however, is lacking. In this study, we examined the physiological role of mPTP opening in renal calcium crystallization in vitro and in vivo. In the in vitro study, cultured renal tubular cells were exposed to calcium oxalate monohydrate (COM) crystals and treated with CsA (2 μM). COM crystals induced depolarization of the mitochondrial membrane potential and generated oxidative stress as evaluated by Cu-Zn SOD and 4-HNE. Furthermore, the expression of cytochrome c and cleaved caspase 3 was increased and these effects were prevented by CsA. In the in vivo study, Sprague-Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with CsA (2.5, 5.0, and 10.0 mg/kg/day) for 14 days. EG administration induced renal calcium crystallization, which was prevented by CsA. Mitochondrial collapse was demonstrated by transmission electron microscopy, and oxidative stress was evaluated by measuring Cu-Zn SOD, MDA, and 8-OHdG generated by EG administration, all of which were prevented by CsA. Collectively, our results provide compelling evidence for a role of mPTP opening and its associated mitochondrial collapse, oxidative stress, and activation of the apoptotic pathway in the initial process of renal calcium crystallization.

Objectives To analyze the changes in the annual incidence and epidemiologic details of lower urinary tract stones in Japan, a nationwide survey of urolithiasis was performed. Methods Data were obtained from all patients who had been diagnosed by urologists in 2005 as having lower urinary tract stones, including both first and recurrent stones. The data were separately enumerated according to hospital size, irrespective of admission and treatment. The study included all hospitals approved by the Japanese Board of Urology and thus covered nearly all urologists practicing in Japan. The estimated annual incidence according to sex, age, and stone composition was compared with other nationwide surveys taken from 1965 to 1995. Results The incidence of lower urinary tract stones in Japan has steadily increased from 4.7/100 000 in 1965 to 9.1/100 000 in 2005. However, the age-standardized annual incidence of lower urinary tract stones in Japan decreased slightly from 5.5/100 000 in 1965 to 5.4/100 000 in 2005. The incidence of stones containing calcium has significantly increased from 50.7% to 72.0% among men and the incidence of infection-related stones has decreased significantly from 26.2% to 10.1%. The ratios of uric acid calculi in men and of infection-related stones in women increased with age. Conclusions The increased incidence of lower urinary tract stones is in slight contrast to the sudden increase in the incidence of upper urinary tract stones, which might be associated with the aging of the Japanese population.

Crystal formation in mice could not be induced either by the administration of ethylene glycol or by glycolate. To clarify the reasons for the difference among these oxalate precursors in mice, we studied renal tubular epithelial injury by immunohistochemical staining of oxidative stress and observing microstructures. Daily intra-abdominal injection of saline solution [10 ml/(kg day)], ethylene glycol[(48.3 mmol/(kg day)], glycolate [1.31 mmol/(kg day)], and glyoxylate [1.35 mmol/(kg day)] into C57BL/6 male mice (8 weeks) was performed for 7 days. Immunohistochemical staining of superoxide dismutase (SOD) and malondialdehyde (MDA), and transmission electron microscopy (TEM) of renal tubular epithelial cells were performed to observe oxidative stress and morphological changes, respectively. Decreased SOD and increased MDA were shown only in glyoxylate-treated mouse kidneys. The TEM study with glyoxylate-treated mouse kidneys demonstrated that the internal structure of mitochondria in renal tubular cells underwent destruction and vacuolization, and microvilli density decreased. These changes in renal tubular cells were located in the crystal-forming area. However, such changes were not detected in the other groups. Each precursor of oxalate induces different changes in renal epithelial cells regarding oxidative stress and the microstructural changes. It is suggested that calcium oxalate crystal formation requires cell injury and morphological changes of renal epithelial tubular cells induced by glyoxylate administration in the mouse kidney.

The establishment of an experimental animal model would be useful to study the mechanism of kidney stone formation. A calcium kidney stone model in rats induced by ethylene glycol has been used for research; however, to investigate the genetic basis affecting kidney stone formation, which will contribute to preventive medicine, the establishment of a kidney stone model in mice is essential. This study indicates the optimum conditions for inducing calcium oxalate stones in normal mouse kidney. Various doses of oxalate precursors, ethylene glycol, glycolate and glyoxylate, were administered either by free drinking or intraabdominal injection for 2 months as a preliminary study. Stone formation was detected with light microscopy, polarized light optical microscopy and electron microscopy. Stone components were detected with X-ray diffraction analysis. The expression of osteopontin (OPN), a major stone-related protein, was detected with immunohistochemical staining, in situ hybridization and quantitative reverse transcriptase polymerase chain reaction. Kidney stones were not detected in ethylene glycol- or glycolate-treated groups even at the highest dose of LD(50). Whereas, numerous kidney stones were detected in glyoxylate-treated mice (more than 60 mg/kg) at 3, 6 and 9 days after glyoxylate were administered intraabdominally. However, the number of kidney stones decreased gradually at day 12, and was hardly detected at day 15. The stone component was further analyzed as calcium oxalate monohydrate. A dramatic increase in the expression of OPN was observed by the administration of glyoxylate. We established a mouse kidney stone experimental system in this study. The difficulty of inducing kidney stones suggested that mice have greater intrinsic ability to prevent stone formation with hyperoxaluric stress than rats. The differing response to hyperoxaluric stress between mice and rats possibly contributes to the molecular mechanism of kidney stone formation and will aid preventive medicine in the future.

We postulated that IgA nephropathy (IgAN) involved alterations of serum IgG. The present study was undertaken to elucidate changes in serum IgG oligosaccharide structure analysis and to assess the diagnostic usefulness of this analysis in IgAN. The subjects were 28 children who were definitively diagnosed as having IgAN on the basis of renal biopsy and who had not received treatment for this disease; 27 healthy children; 15 untreated adults definitely diagnosed as having IgAN; 5 patients with other nephropathies; and 61 healthy adults. Oligosaccharide analyses of IgG were performed by reverse-phase high-performance liquid chromatography (HPLC) developed by Takahashi and colleagues. In both the children and the adults, the peak area ratio of isomers with two different galactosyl-N-acetylglucosamine (Gal-GlcNAc) binding sites was significantly lower in the presence of IgAN than in the healthy subjects (P < 0.05 in children and P < 0.001 in adults). The ratio of Gal-free oligosaccharides to Gal-positive oligosaccharides did not differ according to the presence or absence of IgAN in children or in adults. The analysis of the oligosaccharide structure of serum IgG seems to be useful in diagnosing IgAN.

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

Sirs, A previously healthy 14-year-old boy presented with mild acute renal dysfunction with proteinuria and glucosuria. His past history and family history were unremarkable. The ophthalmologic examinations were normal. The patient was taking no medicines. Blood urea nitrogen of 18 mg/dl, creatinine of 1.3 mg/dl and creatinine clearance of 107.4 ml/min/1.73 m 2 were all measured. An autoantibody screen was negative. Urinary excretion of N-acetyl-beta-glucosaminidase (NAG) was 63.2 U/l (normal 0.3–11.5 U/l), and b2-microglobulin (b2-MG) was 37,079 �g/l (normal 30–340 �g/l). Serological tests for Epstein-Barr virus (EBV) showed the following: EBVviral capsid antigen (VCA) IgG 80x (negative