Masahito Hirose

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.

Morphological conversion of calcium oxalate crystals into stones is regulated by osteopontin in mouse kidney.

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.

Genome‐Wide Analysis of Genes Related to Kidney Stone Formation and Elimination in the Calcium Oxalate Nephrolithiasis Model Mouse: Detection of Stone‐Preventive Factors and Involvement of Macrophage Activity

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.

Efficacy of selective α1A adrenoceptor antagonist silodosin in the medical expulsive therapy for ureteral stones

Recently, we reported that α1A adrenoceptor (AR) is the main participant in phenylephrine‐induced human ureteral contraction. We therefore decided to carry out a prospective randomized study to evaluate the effects of silodosin, a selective α1A AR antagonist, as a medical expulsive therapy for ureteral stones. A total of 187 male patients, who were referred to our department for the management of symptomatic unilateral ureteral calculi of less than 10 mm, were randomly divided into two groups: group A (92 patients), who were instructed to drink 2 L of water daily, and group B (95 patients), who received the same instruction and were also given silodosin (8 mg/daily) for a maximum of 8 weeks. Expulsion rate, mean expulsion time and need for analgesics were examined. Overall, the mean expulsion time was 15.19 ± 7.14 days for group A and 10.27 ± 8.35 days for group B (P = 0.0058). In cases involving distal ureteral stones, the mean expulsion time was 13.40 ± 5.90 and 9.29 ± 5.91 days, respectively (P = 0.012). For stones of 1–5 mm in diameter, the mean expulsion time was 14.28 ± 6.35 and 9.56 ± 8.45 days, respectively (P = 0.017). For stones of 6–9 mm in diameter, the stone expulsion rate was 30.4% and 52.2% (P = 0.036), and the mean expulsion time was 21.00 ± 9.9 and 11.33 ± 8.31 days, respectively (P = 0.038). Herein, we report the first on silodosin in the management of ureteral lithiasis. Our findings suggest that silodosin might have potential as a medical expulsive therapy for ureteral stones.

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.

Renal tubular epithelial cell injury and oxidative stress induce calcium oxalate crystal formation in mouse kidney.

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.

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.

Glyoxylate induces renal tubular cell injury and microstructural changes in experimental mouse

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.

Effect of partial cystectomy on the induction of pre-neoplastic lesions in rat bladder initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine followed by bladder carcinogens and promoters

SummaryThe effect of partial cystectomy on the occurrence of pre-neoplastic lesions, papillary or nodular hyperplasia (PN hyperplasia), of the bladder in male F344 rats was studied in an experiment in which bladder carcinogens and promoters were given to the rats after initiation with BBN. The bladder carcinogens tested were N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) and N-4[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and the bladder promoters were sodium saccharin, sodium cyclamate, and DL-tryptophan. Partial cystectomy significantly decreased the occurrence of PN hyperplasia in rats treated with EHBN and tended to inhibit that in rats given saccharin or tryptophan. Thus partial cystectomy inhibited rather than enhanced the induction of PN hyperplasia.