Novel effect of the inhibitor of mitochondrial cyclophilin D activation, N-methyl-4-isoleucine cyclosporin, on renal calcium crystallization.

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.

A paracrine mechanism involving renal tubular cells, adipocytes and macrophages promotes kidney stone formation in a simulated metabolic syndrome environment.

PURPOSE: We developed an in vitro system composed of renal tubular cells, adipocytes and macrophages to simulate metabolic syndrome conditions. We investigated the molecular communication mechanism of these cells and their involvement in kidney stone formation. MATERIALS AND METHODS: Mouse renal tubular cells (M-1) were cocultured with adipocytes (3T3-L1) and/or macrophages (RAW264.7). Calcium oxalate monohydrate crystals were exposed to M-1 cells after 48-hour coculture and the number of calcium oxalate monohydrate crystals adherent to the cells was quantified. The expression of cocultured medium and M-1 cell inflammatory factors was analyzed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. RESULTS: The inflammatory markers MCP-1, OPN and TNF-α were markedly up-regulated in cocultured M-1 cells. OPN expression increased in M-1 cells cocultured with RAW264.7 cells while MCP-1 and TNF-α were over expressed in M-1 cells cocultured with 3T3-L1 cells. Coculturing M-1 cells simultaneously with 3T3-L1 and RAW264.7 cells resulted in a significant increase in calcium oxalate monohydrate crystal adherence to M-1 cells. CONCLUSIONS: Inflammatory cytokine changes were induced by coculturing renal tubular cells with adipocytes and/or macrophages without direct contact, indicating that crosstalk between adipocytes/macrophages and renal tubular cells was mediated by soluble factors. The susceptibility to urolithiasis of patients with metabolic syndrome might be due to aggravated inflammation of renal tubular cells triggered by a paracrine mechanism involving these 3 cell types.

Increased crystal-cell interaction in vitro under co-culture of renal tubular cells and adipocytes by in vitro co-culture paracrine systems simulating metabolic syndrome.

We established an experimental co-culture system for renal tubular cells and adipocytes to investigate kidney stone formation mechanisms under metabolic syndrome (MetS) conditions and examined the interaction between these cells morphologically and genetically. M-1s and 3T3-L1s were cultured individually (control, CON), with 24-h culture media from each cell type added to the other cell type (replacement, RP) in 2-layer co-culture dishes for 24 h (transwell, TW). M-1s were then exposed to calcium oxalate monohydrate (COM) crystals, and attached (14)C-labeled COM crystals were quantified. Expression of kidney stone- and adipocyte-related genes was analyzed. The radioactivity of adherent COM crystals significantly increased in TW and was relatively higher in RP compared to CON. M-1s demonstrated significant upregulation of adiponectin (Adipoq) in RP and secreted phosphoprotein 1 (Spp1) in TW compared to CON before COM crystal exposure, and significant downregulation of Spp1 in TW and upregulation of tumor necrosis factor (Tnf), interleukin 6 (Il-6), and chemokine (C-C motif) ligand 2 (Ccl2) compared to CON after COM crystal exposure. 3T3-L1s showed significant upregulation of Spp1, Adipoq, Tnf-α, and Ccl2 compared to CON. Enzyme-linked immunosorbent assays of co-culture medium revealed significantly increased TNF-α in TW. Our results highlight the potential for paracrine interactions between renal tubular cells and adipocytes and suggest that MetS conditions may lead to kidney stone formation.

Impact of official technical training for urologists on the efficacy of shock wave lithotripsy.

To evaluate the efficacy of company-initiated training of urologists on shock wave lithotripsy (SWL) treatment results, we retrospectively assessed 602 patients who underwent SWL in Nagoya City University Hospital between January 2004 and June 2011 using Lithotripter S (Dornier MedTech, Japan). Training-provided by a training specialist of the company in June 2010-focused on the targeting of renal and proximal ureter stones with a combination of radiography and ultrasonography (US). The stretcher wedges were positioned in the semi-prone position or the semi-supine position for middle and distal ureter stones, respectively. Success rates between 519 pre-training treatments and 83 post-training treatments were compared. Patient age and stone location, burden, number, and composition did not significantly differ between pre- and post-training. Training improved the overall success rate from 66.3 to 87.2 % (P < 0.0001). The mean number of SWL treatments decreased from 1.8 ± 1.8 to 1.4 ± 1.3 (P = 0.01). The first SWL treatment success rate increased from 67.1 to 83.7 % (P = 0.002), and the need for multiple treatments decreased. The frequency of detection of renal and proximal ureter stones by both radiography and US increased from 10.5 % before training to 58.2 % after training (P < 0.0001). Significant factors for successful SWL were determined to be training and prone position for distal ureter stones by multivariate analysis and ultrasonic detection for renal and proximal ureter stones by univariate analysis. Skills in targeting stones using ultrasonography and selecting the proper therapeutic position are essential for improving the success rate of stone removal.

Oxygen nano-bubble water reduces calcium oxalate deposits and tubular cell injury in ethylene glycol-treated rat kidney.

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-ß-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.

Effect of adiponectin on kidney crystal formation in metabolic syndrome model mice via inhibition of inflammation and apoptosis.

The aims of the present study were to elucidate a possible mechanism of kidney crystal formation by using a metabolic syndrome (MetS) mouse model and to assess the effectiveness of adiponectin treatment for the prevention of kidney crystals. Further, we performed genome-wide expression analyses for investigating novel genetic environmental changes. Wild-type (+/+) mice showed no kidney crystal formation, whereas ob/ob mice showed crystal depositions in their renal tubules. However, this deposition was remarkably reduced by adiponectin. Expression analysis of genes associated with MetS-related kidney crystal formation identified 259 genes that were >2.0-fold up-regulated and 243 genes that were <0.5-fold down-regulated. Gene Ontology (GO) analyses revealed that the up-regulated genes belonged to the categories of immunoreaction, inflammation, and adhesion molecules and that the down-regulated genes belonged to the categories of oxidative stress and lipid metabolism. Expression analysis of adiponectin-induced genes related to crystal prevention revealed that the numbers of up- and down-regulated genes were 154 and 190, respectively. GO analyses indicated that the up-regulated genes belonged to the categories of cellular and mitochondrial repair, whereas the down-regulated genes belonged to the categories of immune and inflammatory reactions and apoptosis. The results of this study provide compelling evidence that the mechanism of kidney crystal formation in the MetS environment involves the progression of an inflammation and immunoresponse, including oxidative stress and adhesion reactions in renal tissues. This is the first report to prove the preventive effect of adiponectin treatment for kidney crystal formation by renoprotective activities and inhibition of inflammation and apoptosis.

Biomolecular mechanism of urinary stone formation involving osteopontin.

Urinary stones consist of two phases-an inorganic (mineral) phase and an organic (matrix) phase. Studies on the organic components of kidney stones have been undertaken later than those on the inorganic components. After osteopontin was identified as one of the matrix components, the biomolecular mechanism of urinary stone formation became clearer. It also triggered the development of new preventive treatments. Osteopontin expression is sporadically observed in normal distal tubular cells and is markedly increased in stone-forming kidneys. Calcium oxalate crystals adhering to renal tubular cells are incorporated into cells by the involvement of osteopontin. Stimulation of crystal-cell adhesion impairs the opening of mitochondrial permeability transition pores (mPTP) in tubular cells and produces oxidative stress, apoptosis, and osteopontin expression. Macrophages phagocytose and digest a small amount of crystals, but many crystals aggregate into a mass containing osteopontin and epithelial cell debris and are excreted into the renal tubular lumen, becoming nuclei of urinary stones. This biomolecular mechanism is similar to atherosclerotic calcification. Based on these findings, new preventive treatments have been developed. Dietary control such as low-cholesterol intake and the ingestion of antioxidative foods and vegetables have successfully reduced the 5-year recurrence rate. Osteopontin antibodies and cyclosporine A, which blocks the opening of mPTP, have markedly inhibited the expression of osteopontin and urinary stone formation in animal models.

Mitochondrial permeability transition pore opening induces the initial process of renal calcium crystallization.

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.

[New therapy using bisphosphonate for urolithiasis].

Osteoporosis is characterized by a reduction in bone mineral density (BMD) . Reduced BMD has been reported in urolithiasis patients with hypercalciuria, as well as in those with normocalciuria. Bisphosphonates potently inhibit bone resorption and are used in the management of osteoporosis. We show the ability of bisphosphonate to prevent the recurrence of urolithiasis. Bisphosphonate reduced the excretion of urinary calcium and the ion activity product index of calcium phosphate in urolithiasis patients, and prevented urinary stone formation in long-term bed rest test. The results suggest that ALN not only improves osteoporosis but also reduces the risk of calcium stone formation. Bisphosponates are believed to reduce the urinary excretion of calcium by improvement of bone metabolism, and to have a direct effect in the prevention of urolithiasis.

[Medical expulsive therapy facilitated by alpha 1 adrenoceptor antagonist].

In 2002, speedy elimination of ureterolithiasis in the lower part of ureter was first reported with the alpha 1 blocker. Thereafter, there are a lot of reports including meta-analysis about tamsulosin. In 2011 EAU Guidelines on Urolithiasis, it is the most important to establish effective MET (medical expulsive therapy) to facilitate spontaneous stone passage. Alpha 1 blockers are the preferred agents for MET. As a basic evidence for MET, we reported that alpha 1a and 1d AR subtype mRNA was highly expressed in the human ureter and that alpha 1A AR is the main participant in the human ureteral contraction. It is published newly in Japanese Guidelines on Urolithiasis revised edition to schedule to be published soon.

The role of long-term loading of cholesterol in renal crystal formation.

We studied the effects of cholesterol load on urinary stone in rats receiving a standard diet or a high fat diet. Sixty male rats were randomized to two groups and were fed either a standard diet (SD group) or a high fat diet (HFD group) for 8 weeks. Then the two groups were further divided into four groups. SD group, HFD group, SD + EG group (with standard diet + ethylene glycol administration for two weeks), and HFD + EG group (with high fat diet + ethylene glycol administration). The starting date of EG administration was considered to be week 0. Twenty-four-hour urine samples were collected in week 0, week 1, and week 2, and oxalate excretion and citrate excretion were measured by capillary electrophoresis analyzer The excretion of phosphorus, magnesium, and creatinine for 24 hours was measured using an automated analyzer Serum sodium, potassium, chloride, calcium, phosphate, magnesium, creatinine, total cholesterol, triglyceride, HDL-cholesterol and glucose were determined using an automated analyzer The kidney tissues were obtained to perform hematoxyline-eosine staining and Pizzolato's staining to detect oxalate-containing crystals. The average body weight in HFD groups and HFD + EG group in week 0 was significantly higher than that of SD group and SD + EG group. The calcium oxalate crystal deposition was not observed in all groups in week 0. HFD + EG group in week 1 had sporadically calcium oxalate crystal deposition in renal distal tubular cells and tubular lumens. In week 2, the number of crystal deposition in HFD + EG group was increased remarkably. The crystals were slightly observed in SD + EG group in week 2. The excretion of urinary calcium and phosphate in HFD group and HFD + EG group was significantly higher than that of the SD group and SD + EG group in week 0. The amount of urinary citrate excretion in the SD group and SD + EG group showed a significantly higher value compared with that of the HFD group and HFD + EG group in week 0. The level of serum total cholesterol in the HFD group and HFD + EG group was higher compared to that in the SD group and SD + EG group. The serum triglyceride level was not significantly different in the four groups in week 0. Interestingly, the level of triglyceride of EG administration groups (SD + EG and HFD + EG group) was significantly higher than that in EG no-administration groups (SD group and HFD group) in week 1 and week 2. The serum glucose level in the HFD group and HFD + EG group was significantly higher than that in the SD group and SD + EG group in week 0. In week 2, the glucose level of EG administration groups (HDF + EG group and SD + EG group) was significantly lower than that of EG no-administration groups (HFD group and SD group). In conclusion, this result suggested that long-term loading of cholesterol could increase renal calcium stone formation.

[Functional domains of osteopontin stimulate renal crystal formation: analysis of OPN-transgenic mice].

Osteopontin (OPN) has been described to play a nonredundant role in the formation of renal crystals. This biological activity of OPN may be attributed to its characteristic structure, which includes 2 calcium binding sites, Arg-Gly-Asp (RGD) sequences. To test this hypothesis, we evaluated wild-type mice (WT group), OPN-knockout mice (KO group), and two types of transgenic mice : (1) one type carrying a transgene in which the sequences coding for the 2 calcium-binding sites of the OPN were deleted (CaX group) and (2) the other type carrying a transgene in which the sequence that codes for the RGD sequence of the OPN was modified to one that codes for Arg-Gly-Glu (RGE ; RGE group). Changes occurring after intraperitoneal injection of glyoxylate for 9 d were analyzed. The amount of crystals deposited was the greatest in mice of the WT group and the least in those of the KO group. The number of crystal deposits in mice of the RGE and KO groups was approximately the same. Microscopic observations revealed that the crystal nuclei in mice in the CaX group were stratified and exhibited a disordered pattern ; this pattern was dissimilar to that observed in the mice in the WT and RGE groups, wherein the crystal nuclei exhibited a rosette petal-like radial pattern. The results indicate the possibility that each domain contributes to the mechanism by which OPN stimulates crystal formation.

Renal macrophage migration and crystal phagocytosis via inflammatory-related gene expression during kidney stone formation and elimination in mice: Detection by association analysis of stone-related gene expression and microstructural observation.

Mice have a strong ability to eliminate renal calcium oxalate crystals, and our previous examination indicated a susceptibility in which monocyte-macrophage interaction could participate in the phenomenon. To clarify the macrophage-related factors playing roles in the prevention of crystal formation in mouse kidneys, morphologic and expression studies based on microarray pathway analysis were performed. Eight-week-old male C57BL/6N mice were administered 80 mg/kg of glyoxylate by daily intraabdominal injection for 15 days, and the kidneys were extracted every 3 days for DNA microarray analysis. Based on the raw data of microarray analysis, pathway analyses of inflammatory response demonstrated macrophage activation through the increased expression of chemokine (C-X-C) ligand 1, fibronectin 1, and major histocompatability (MHC) class II. Association analysis of related gene expression values by quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated the high association of chemokine (C-C) ligand 2, CD44, colony-stimulating factor 1, fibronectin 1, matrix gla protein, secreted phosphoprotein 1, and transforming growth factor ß1 (TGF-ß1) with the amount of both renal crystals and F4/80, a macrophage marker. Immunohistochemically, interstitial macrophages increased during the experimental course, and CD44 and MHC class II were upregulated around crystal-formation sites. Ultrastructural observation of renal macrophages by transmission electron microscopy indicated interstitial macrophage migration with the phagocytosis of crystals. In conclusion, increased expression of inflammation-related genes of renal tubular cells induced by crystal formation and deposition could induce monocyte-macrophage migration and phagocytosis via the interaction of CD44 with osteopontin and fibronectin. Such crystal-removing ability of macrophages through phagocytosis and digestion might become a new target for the prevention of stone formation.

The mechanism of renal stone formation and renal failure induced by administration of melamine and cyanuric acid.

Renal stone formation and renal failure among Chinese infants administered melamine-containing formula were increasingly reported in 2008. We investigated the mechanism by which melamine and cyanuric acid induce renal stone formation and renal failure. Ten-week-old rats were administered either melamine [2.4, 24, or 240 mg/kg/day], both melamine and cyanuric acid [each at 1.2, 12, or 120 mg/kg/day], or water (controls). Blood and 24-h urine samples and kidney sections were evaluated on days 3, 7, and 14. In rats administered melamine alone or the low-dose melamine/cyanuric acid combination [1.2 mg/kg/day], crystals were not detected. On day 3, crystal formation was observed in the renal distal tubular lumens and collecting ducts of rats administered the intermediate-dose melamine/cyanuric acid [12 mg/kg/day], and the number of crystals increased during the course of the experiment. In rats administered the high-dose melamine/cyanuric acid [120 mg/kg/day], crystals were found in the proximal tubular lumens of the renal cortex on day 3, but acute renal failure resulted in death by day 7. Polarized light optical microphotography and scanning electron microscopy revealed tubular lumens occluded by a layer of axle-shaped crystals. X-ray diffraction findings revealed a nitrogen component but no calcium. The upper regions of occluded tubes were expanded, and the epithelium was thin. Melamine and cyanuric acid in combination, but not by melamine alone induce crystal formation and affected renal functioning. Renal failure due to melamine cyanurate crystals appears to occur via tubular occlusion.

Embryonal rhabdomyosarcoma of the prostate.

We describe a case of embryonal rhabdomyosarcoma of the prostate in a 20-year-old man with a history of acute lymphatic leukemia at 6 years of age. He presented with gross hematuria and high fever. The level of prostate-specific antigen (PSA) was 27.9 ng/ml. Computed tomography revealed a suspected infectious cyst in the prostate. After antibiotic treatment was begun, the PSA level normalized. However, the patient later suffered bladder tamponade. A biopsy of the prostate revealed embryonal rhabdomyosarcoma. Chemotherapy with vincristine, actinomycin D, cyclophosphamide, and ifosfamide was begun according to the treatment protocol of the Japan Rhabdomyosarcoma Study Group. The tumor shrank, and partial remission was obtained after 1 course of chemotherapy. During treatment for acute lymphatic leukemia at the age of 6 years, the patient had been exposed to a cumulative radiation dose of 10 Gy across his entire body. It has been reported that 88% of postradiation sarcomas are KIT-positive, and we suspect that our patient suffered a postradiation sarcoma, because his tumor was KIT-positive. This is the first report of postradiation sarcoma manifesting as an embryonal rhabdomyosarcoma of the prostate.

A case of oncocytic papillary renal cell carcinoma.

Papillary renal cell carcinomas (RCC) are the second most frequently identified pathological subtypes of RCC. Occasionally, papillary RCC demonstrate pathological characteristics of renal oncocytomas (RO), benign renal tumors. We report the case of an 81-year-old woman with an oncocytic papillary RCC, which was difficult to differentiate from a hybrid of RO and papillary RCC, who underwent left radical nephrectomy. Morphological examination showed oncocytic tumor region and partially scattered regions with papillary structure. Immunohistochemical examinations demonstrated strongly positive staining of alpha-methylacyl-CoA racemase in the papillary region and negative staining of progesterone receptor and CD117 in both regions. Fluorescence in situ hybridization confirmed the increased number of copies of chromosome 7 in the papillary region. Comprehensively, this case could be diagnosed as oncocytic papillary RCC. No evidence of disease recurrence was found at 12 months' follow up.