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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.

INTRODUCTION AND OBJECTIVES: We recently reported that renal tubular cell injury induced by oxidative stress and subsequent mitochondrial collapse is the initial step in the process of kidney stone formation. Recently, it was shown that the mitochondrial permeability transition (MPT) is involved in mitochondrial collapse. Cyclophilin D (CypD) is a mitochondrial matrix protein involved in MPT. Thus, we hypothesized that CypD-dependent MPT causes mitochondrial collapse and is a trigger of kidney stone formation. In this study, we investigated whether mitochondrial collapse depends on CypD and determined its relationship to kidney stone formation using CypD-deficient mice. METHODS: We administered 80 mg$kg 1 glyoxylic acid, a precursor of oxalic acid, intraperitoneally for 6 consecutive days to 8week-old male CypD-deficient mice (CypD-/-, n 1⁄4 6) and wild-type mice (CypDþ/þ, n 1⁄4 6). We removed the kidneys of the mice in each group 6 days after glyoxylic acid administration. Stone formation and morphology were evaluated using a polarization microscope and Pizzolato staining, and the stone formation rate was estimated using image analysis software. The form of mitochondria was observed using a transmission electron microscope (TEM). Superoxide dismutase (SOD) and malondialdehyde (MDA), markers of oxidative stress, osteopontin (OPN), a stone matrix protein, and caspase 3, a marker of apoptosis, were evaluated by immunohistochemical staining and western blotting. RESULTS: The stone formation rate of CypD-/mice (0.05%) was significantly lower than that of CypDþ/þ mice (0.22%). Expression of MDA, OPN, and caspase 3 was lower in CypD-/mice than in CypDþ/þ mice. Expression of SOD was higher in CypD-/than in CypDþ/þ mice. Based on TEM observations, CypDþ/þ mice had a disorganized internal structure and rupture of the double membrane of the mitochondria suggesting mitochondrial collapse, but such morphological changes were rare in CypD-/mice. CONCLUSIONS: According to the results of this study, during kidney stone formation, CypD influences MPT by generating mitochondrial collapse via oxidative stress, leading to renal tubular cell injury and kidney stone formation. We elucidated the mechanism of CypDdependent MPT in promoting kidney stone formation.

INTRODUCTION AND OBJECTIVES: Renal tubular cell injury induced by oxalate plays an important role in kidney stone formation. Water containing oxygen nano-bubbles (ONB: nanometersized bubbles generated from oxygen micro-bubbles) has anti-inflammatory effects. Therefore, we investigated the inhibitory effects of ONB water on kidney stone formation in ethylene glycol (EG)-treated rats. METHODS: ONB water can be produced by rapidly collapsing oxygen micro-bubbles in hard water with a salt concentration of 1.0% by mass. Further, ONB water with a salt concentration of 0% by mass can be prepared by passing the resulting ONB water twice through a reverse osmosis membrane, obtaining ONB water was defined as 100% ONB water. A mixture comprising half 100% ONB water and half distilled water was defined as 50% ONB water. We divided 60 rats, aged 4 weeks, into 5 groups: (1) control, a water-fed group; (2) 100% ONB, a 100% ONB water-fed group; (3) EG, the EG-treated water-fed group; and (4), and (5) EG+50% ONB and EG+100% ONB, the 2 EGtreated, ONB water-fed groups. The rats consumed these fluids by free access for 5 weeks, and the EG-treated groups were also administered 1.2 mL of 10% EG and 0.5 mg of vitamin D3 via a stomach tube for the last 2 weeks. Six rats in each group were sacrificed after 7 days and 6 after 14 days. The pooled 24-hour urine and blood samples of these rats were collected, and their kidneys were excised. Renal CaOx deposits, urinary excretion of N-acetyl-b-D-glucosaminidase (NAG), and renal expression of MCP-1 and OPN as inflammation-related proteins, and the crystal binding molecule hyaluronic acid were compared among the 5 groups. RESULTS: In the EG group, CaOx deposits tended to increase at 14 days compared to 7 days after EG administration. However, CaOx deposits were significantly lower in the EG+50% ONB and EG+100% ONB groups than in the EG group 14 days after administration (Figure). In the EG+50% ONB and EG+100% ONB groups, ONB water significantly decreased urinary NAG excretion, and renal MCP-1, OPN, and hyaluronic acid expression compared to the EG group. CONCLUSIONS: ONB water exerted substantial effects on kidney stone formation in the rat kidney by inhibiting renal tubular cell injury. ONB water may be a prophylactic for kidney stones.

the kidneys were harvested and examined calcium oxalate stones/crystals were suspected. Histopathological analysis on H&E stainings, fluorescent microscopy and stone analysis by infrared spectrum analysis confirmed calcium oxalate stones/crystal formation within the renal cortex and medulla. No crystal deposition was observed in the control group. CONCLUSIONS: Pigs fed ethylene glycol in combination with vitamin D, ammonium chloride, gentamycin, or Lasix formed calcium oxalate stones/crystals. The use of the above feeding substrate is inexpensive and effective at producing a novel porcine calcium oxalate stone forming model.

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.