Iwao Yoshioka

Atorvastatin Inhibits Renal Crystal Retention in a Rat Stone Forming Model

PURPOSE The interactions between crystals and renal tubular cells are important factors in urolithiasis formation. Moreover, some reports have suggested the involvement of renal tubular cell injury in crystal-cell interaction processes. Atorvastatin, which is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A, is prescribed to decrease high cholesterol levels, and it has anti-inflammation and anti-oxidization activities. Atorvastatin is also reported to control transforming growth factor-beta1 expression. We investigated whether atorvastatin can prevent renal tubular cell injury by oxalate and inhibit renal crystal retention. MATERIALS AND METHODS Ten-week-old specific pathogen-free male Sprague-Dawley rats were used. Atorvastatin (2 mg/kg) in 0.5% carboxymethyl cellulose was administered orally daily for 2 weeks. The rats were separated into 4 experimental groups, including group 1--water and 0.5% carboxymethyl cellulose daily, group 2--water and atorvastatin in 0.5% carboxymethyl cellulose daily, group 3--1% ethylene glycol dissolved in water, 0.5 mug vitamin D3 dissolved in 1 ml salad oil and 0.5% carboxymethyl cellulose daily, and group 4--1% ethylene glycol dissolved in water, 0.5 microg vitamin D3 dissolved in 1 ml salad oil and atorvastatin in 0.5% carboxymethyl cellulose daily. The ethylene glycol model of hyperoxaluria and the effect of atorvastatin treatment were analyzed in groups 1 to 4. Urine samples were collected every 24 hours in metabolic cages and analyzed immediately or stored at -70C until analysis. The rats were sacrificed after 2 weeks and the kidneys were removed for further examination. We measured urinary N-acetyl glucosaminidase levels as a biomarker of renal tubular cell injury and urinary 8-OHdG as a biomarker of oxidative stress in 24-hour urine samples. Removed kidneys were used for quantitative analysis of the superoxide dismutase level and the detection of apoptosis. Finally, we measured the amount of crystal deposits in renal tubular cells. RESULTS Urinary N-acetyl glucosaminidase and 8-OHdG levels were decreased significantly by atorvastatin treatment in this stone forming rat model. Atorvastatin treatment increased the superoxide dismutase level and inhibited the degree of renal tubular cell N-acetyl glucosaminidase compared with stone forming control group 3. A decrease in renal crystal retention was noted when excised kidneys were evaluated following atorvastatin treatment. CONCLUSIONS Atorvastatin was found to have inhibitory effects on the renal tubular cell injury and oxidative stress caused by oxalate and crystals. Atorvastatin inhibited renal crystal retention. We believe that atorvastatin could help prevent and treat renal crystal formation.

Effects of flutamide as a second‐line agent for maximum androgen blockade of hormone refractory prostate cancer

Abstract:  We analyzed clinical effects of flutamide as a second‐line agent for maximum androgen blockade (MAB) in patients with relapsing prostate cancer who received bicalutamide as the first‐line MAB agent. This study included 13 patients with progressive prostate cancer who had relapsed after first‐line MAB, with bicalutamide at 80 mg/day. After checking for antiandrogen withdrawal syndrome, they were given flutamide at 375 mg/day as second‐line MAB. The effectiveness of that therapy was evaluated by changes in prostatic specific antigen (PSA) levels, with response defined as a decrease of greater than 50% from the start of therapy. We also compared several factors between responders and non‐responders. Nine (69.2%) of the 13 patients showed a decrease in PSA levels, of whom five (38.5%) had a greater than 50% decrease and were defined as responders. The median duration of PSA response was 11.0 months (range 5–20 months). Patients who had a longer duration of response to first‐line MAB had a significantly greater response to second‐line MAB. For advanced prostate cancer patients who progressed on first‐line MAB with bicalutamide, flutamide administration as a second‐line antiandrogen was found to be relatively effective, especially for those who showed a longer duration of response to the first‐line MAB. Our results confirm previous findings that MAB using flutamide is an effective second‐line hormonal therapy.

Effect of sex hormones on crystal formation in a stone-forming rat model.

OBJECTIVES To evaluate the contribution of sex hormones in urolithiasis using a stone-forming rat model. Gender differences because of sex hormones are thought to influence the incidence of urolithiasis. METHODS We divided rats into 7 groups, such as intact males, orchidectomized males, intact males subcutaneously implanted with testosterone, intact males subcutaneously implanted with estradiol, intact females, ovariectomized females, and intact females subcutaneously implanted with testosterone. At 10 weeks old, the rats were fed 0.5% ethylene glycol in drinking water and given 0.5 microg of 1,25-dihydroxy vitamin D(3). Kidney crystal deposition and the degree of oxidative stress were examined in each group, and endogenous oxalate metabolism and antioxidant enzymes were compared among groups using real-time reverse transcription-polymerase chain reaction. RESULTS Extensive crystal deposition was observed in intact males and testosterone-administered males, whereas few crystals were found in intact females. Crystal deposition was inhibited in orchiectomized males and in those administered estradiol, whereas ovariectomized females and testosterone-administered females had slightly enhanced and very enhanced crystal deposition, respectively. Increases in urinary oxalate excretion paralleled renal crystal deposition, which were both enhanced by testosterone treatment through increased glycolate oxidase expression. Oxidative stress increased in groups with extensive crystal deposition compared with those without. Antioxidant enzyme expression was enhanced by estradiol. CONCLUSIONS Testosterone was a promoter and estradiol an inhibitor of kidney crystal deposition, likely because of their effects on oxalate synthesis and oxidative stress.

Why does atorvastatin inhibit renal crystal retention

Recently, we reported that atorvastatin prevents renal tubular cell injury by oxalate and inhibits renal crystal retention. In this study, we investigated the mechanism by which atorvastatin inhibits renal crystal retention. Male Sprague-Dawley rats were separated into four experimental groups, and the ethylene glycol model of hyperoxaluria and the atorvastatin treatment model were analyzed. To clarify the mechanism by which atorvastatin inhibits renal crystal retention, the removed kidneys were used for the quantitative analysis of superoxide dismutase (SOD) and catalase. The subunits of the NADPH oxidase system were evaluated using real-time polymerase chain reaction analysis. Furthermore, the level of transforming growth factor-β (TGF-β) in kidney tissue was compared in each group. Atorvastatin treatment increased the SOD and catalase level compared with the stone-forming control group. Atorvastatin treatment decreased the expression of NOX-1 mRNA. Furthermore, the level of TGF-β was suppressed by atorvastatin treatment. We found that atorvastatin have inhibited calcium oxalate (CaOX) urolithiasis formation. We hypothesize that the mechanism of action of atorvastatin involves inhibiting TGF-β and NADPH oxidase, and increasing the SOD and catalase level. We believe that atorvastatin will be helpful in the treatment of CaOX urolithiasis.

Angiotensin Type-1 Receptor Blocker Candesartan Inhibits Calcium Oxalate Crystal Deposition in Ethylene Glycol-Treated Rat Kidneys

OBJECTIVES To investigate whether an angiotensin type-1 receptor blocker could inhibit calcium oxalate crystal deposition using ethylene glycol-treated rats. The renoprotective effect has been reported to be another role of angiotensin type-1 receptor blockers in addition to their role in lowering blood pressure. Recent research has suggested that inhibiting reactive oxidative species generation and tubulointerstitial inflammation is the major role of angiotensin type-1 receptor blockers. These 2 factors are also important in the mechanism of calcium oxalate stone formation. METHODS We divided 28 rats, aged 7 weeks, into 4 groups: group 1, control rats; group 2, candesartan-treated rats; group 3, stone-forming rats; and group 4, candesartan-treated stone-forming rats. The kidney crystal deposits were examined, and the oxidative stress biomarker, nicotinamide adenine dinucleotide phosphate oxidase activity, general and urinary variables, and the transforming growth factor-β level in kidney tissue were compared among the 4 groups. RESULTS The candesartan-treated rats were healthy and had weight gain similar to that of the control rats, although a significant reduction in blood pressure was observed. The urinary components associated with calcium oxalate stone formation were not influenced by candesartan treatment; however, significantly fewer crystal deposits were observed in group 4. The oxidative biomarker and nicotinamide adenine dinucleotide phosphate oxidase activity decreased, and the level of transforming growth factor-β was suppressed in group 4. CONCLUSIONS Candesartan had substantial effects on crystal formation in the rat kidney by suppressing nicotinamide adenine dinucleotide phosphate oxidase and the transforming growth factor-β levels.

MiR-21-5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinoma

Background Extracellular vesicles are lipid bilayer vesicles containing protein, messengerRNA and microRNA. Cancer cell-derived extracellular vesicles may be diagnostic and therapeutic targets. We extracted extracellular vesicles from urine of urothelial carcinoma patients and the control group to identify cancer-specific microRNAs in urinary extracellular vesicles as new biomarkers. Materials and methods microRNA from urinary extracellular vesicles extracted from 6 urothelial carcinoma patients and 3 healthy volunteers was analyzed. We verified candidate microRNAs in an independent cohort of 60 urinary extracellular vesicles samples. To normalize the microRNA expression level in extracellular vesicles, we examined the following in extracellular vesicles: protein concentration, CD9 intensity, amounts of whole miRNAs, RNA U6B small nuclear expression and the creatinine concentration of original urine correlating with the counts of extracted extracellular vesicles measured by the NanoSight™ system. RESULTS From the microarray results 5 microRNAs overexpressed in urinary extracellular vesicles of urothelial carcinoma patients were identified. Creatinine concentration of original urine correlated most with particle counts of extracellular vesicles, indicating that creatinine could be a new tool for normalizing microRNA expression. MiR-21-5p was the most potent biomarker in urinary extracellular vesicles (sensitivity, 75.0%; specificity, 95.8%) and was also overexpressed in urinary extracellular vesicles from urothelial carcinoma patients with negative urine cytology. For the subgroup with negative urine cytology, the sensitivity was 75.0% and specificity was 95.8%. Conclusion MiR-21-5p in urinary extracellular vesicles could be a new biomarker of urothelial carcinoma, especially for urothelial carcinoma patients with negative urine cytology.

Diversity in Protein Profiles of Individual Calcium Oxalate Kidney Stones

Calcium oxalate kidney stones contain low amounts of proteins, some of which have been implicated in progression or prevention of kidney stone formation. To gain insights into the pathophysiology of urolithiasis, we have characterized protein components of calcium oxalate kidney stones by proteomic approaches. Proteins extracted from kidney stones showed highly heterogeneous migration patterns in gel electrophoresis as reported. This was likely to be mainly due to proteolytic degradation and protein-protein crosslinking of Tamm-Horsfall protein and prothrombin. Protein profiles of calcium oxalate kidney stones were obtained by in-solution protease digestion followed by nanoLC-MALDI-tandem mass spectrometry, which resulted in identification of a total of 92 proteins in stones from 9 urolithiasis patients. Further analysis showed that protein species and their relative amounts were highly variable among individual stones. Although proteins such as prothrombin, osteopontin, calgranulin A and calgranulin B were found in most stones tested, some samples had high contents of prothrombin and osteopontin, while others had high contents of calgranulins. In addition, calgranulin-rich stones had various neutrophil-enriched proteins such as myeloperoxidase and lactotransferrin. These proteomic profiles of individual kidney stones suggest that multiple systems composed of different groups of proteins including leucocyte-derived ones are differently involved in pathogenesis of individual kidney stones depending on situations.

Finger taps and constipation are closely related to symptoms of overactive bladder in male patients with Parkinson's disease.

To assess which motor and non‐motor symptoms are closely related to overactive bladder severity in male patients with Parkinsons disease.

Mechanism Underlying the Low Prevalence of Pediatric Calcium Oxalate Urolithiasis

PURPOSE Urinary macromolecules in children show stronger inhibition of Ca oxalate crystal growth, aggregation and adhesion than in adults. To investigate the mechanism of Ca oxalate urolithiasis we evaluated the differences in inhibitory activity against oxalate induced renal cell injury between adults and children. MATERIALS AND METHODS Urine samples were collected from healthy men and their sons. The protective effects of urinary macromolecules against oxalate induced injury to Madin-Darby canine kidney cells (ATCC) were examined by lactate dehydrogenase assay and immunostaining. Variations in the relative abundance of proteins involved in stone formation, such as osteopontin and calgranulin B, were analyzed. RESULTS The urine of children had significantly higher urinary macromolecule and glycosaminoglycan concentrations than that of adults (p <0.01). Urinary macromolecules inhibited oxalate induced Madin-Darby canine kidney cell injury in a concentration dependent manner and stronger activity was observed in children (p <0.05). TUNEL staining and 8-OHdG immunostaining indicated stronger inhibition of apoptosis and oxidative stress in Madin-Darby canine kidney cells pretreated with pediatric urinary macromolecules (p <0.01). Osteopontin and calgranulin B expression correlated positively (p = 0.03). These proteins showed greater down-regulation in children (p <0.01). Osteopontin expression also correlated positively with lactate dehydrogenase release (p = 0.03). CONCLUSIONS A reason for the low prevalence of pediatric urolithiasis is that pediatric urinary macromolecules have stronger inhibitory effects against oxalate induced renal cell injury and oxidative stress induced apoptosis. Furthermore, results suggest that osteopontin and calgranulin B expression is down-regulated in children due to this inhibitory effect and, thus, stone nidus formation is controlled.

Primary Screening of Single Nucleotide Polymorphisms in Human Calreticulin 3 (CALR3)

Calreticulin 3 (CALR3) is considered a candidate gene in human male infertility because male, but not female, CALR3-deficient mice are infertile. To investigate the possible association between variations in CALR3 and impaired spermatogenesis in humans, we screened for mutations in human CALR3 using DNA from 892 infertile male patients and 167 proven-fertile male volunteers. The frequent appearance of several single nucleotide polymorphisms (SNPs), including 742G>T (Asp248Tyr), 976G>A (Asp326Asn), and 1058A>T (Lys353Met), were found in the infertile group by direct sequencing of amplified fragments using the same primers as for polymerase chain reaction. Our results indicate that three major SNPs associated with male infertility exist in the open reading frame of CALR3.