Kazuhiro Niimi

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 β1 (TGF‐β1) 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.

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.

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.

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

To experimentally evaluate the clinical application of N‐methyl‐4‐isoleucine cyclosporin, a novel selective inhibitor of cyclophilin D activation.

MP33-15 MITOCHONDRIAL COLLAPSE DEPENDS ON CYCLOPHILIN D IN RENAL TUBULAR CELLS PROMOTES KIDNEY STONE FORMATION

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

MP25-07 DEVELOPMENT OF A NEW BIOMARKER AND TREATMENT STRATEGY FOR KIDNEY STONE DISEASE TARGETING MITOCHONDRIAL CYCLOPHILIN D ACTIVITY

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.

2303 BISPHOSPHONATE PREVENTS UROLITHIASIS IN MEN WITH OSTEOPOROSIS BY REDUCING THE URINARY ION ACTIVITY PRODUCT OF CALCIUM STONE

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.

2087 CRYSTAL KINETICS AND PROCESSING IN HUMAN KIDNEY STONE FORMATION: COMPARISON OF CLINICAL AND PATHOLOGICAL FINDINGS IN STONE FORMERS AND NON-STONE FORMERS

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.

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

by renal macrophages was observed under a transmitted electron microscope (TEM). RESULTS: Experiment 1: J774.1 indicated higher COM crystal phagocytosis rate than M1 (p 0.0038). The rate increased along with the increase in exposure to the COM crystals (COM, 12.5 g/cm: 19.3 5.86%; COM, 62.5 g/cm: 39.7 5.51%). The phagocytosis rates in the CB groups were significantly lower than those in the non-CB groups (COM, 12.5 g/cm: 0.7 1.15%, p 0.0056; COM, 62.5 g/cm: 13.3 3.21%, p 0.0020). Experiment 2: The number of renal tubular crystals increased until day 6 and thereafter decreased and disappeared until day 15. Renal macrophages appeared in the interstitium, and their number increased or decreased, correlating with the amount of crystals. The TEM image demonstrated phagocytosis of the crystals by interstitial macrophages. CONCLUSIONS: Macrophages have the ability to englobe calcium oxalate crystals and thus play an active role in preventing kidney stone formation.