Nanobacteria have been isolated from kidney stones and it has been suggested that they may act as a nucleus for the initiation of the renal stones. In the present study, we examine their role in biocrystallization and their in vivo effects on kidney pathology. Calcium oxalate monohydrate (COM) assay was carried out in the presence of nanobacteria to study biocrystallization. Wistar rats were given an intravenous injection of nanobacteria and the kidneys were examined for pathological changes. The COM assay showed accelerated biocrystallization of (14)C-oxalate in the presence of nanobacteria, indicating them to be efficient candidates for biomineralization. Histopathological studies revealed bacteria induced renal tubular calcifications and various manifestations of infection. Our studies confirm that nanobacteria may be involved in the pathogenesis of renal tubular calcification.

Calcifying biologic nanoparticles (NPs) develop under cell culture conditions from homogenates of diverse tissue samples displaying extraosseous mineralization, including kidney stones and calcified aneurysms. Probes to definitively identify NPs in biologic systems are lacking. Therefore, the aim of this study was to begin to establish a proteomic biosignature of NPs in order to facilitate more definitive investigation of their contribution to disease. Biologic NPs derived from human kidney stones and calcified aneurysms were completely decalcified by overnight treatment with EDTA or brief incubation in HCl, as evidenced by lack of a calcium shell and of Alizarin Red S staining, by transmission electron microscopy and confocal microscopy, respectively. Decalcified NPs contained numerous proteins including some from bovine serum and others of prokaryotic origin. Most prominent of the latter group was EF-Tu, which appeared identical to EF-Tu from S. epidermidis. A monoclonal antibody against human EF-Tu recognized a protein in Western blots of total NP lysate, as well as in intact NPs by immunofluorescence and immunogold EM. Approximately 8% of NPs were quantitatively recognized by the antibody by flow cytometry. Therefore, we have defined methods to reproducibly decalcify biologic NPs, and identified key components of their proteome. These elements, including EF-Tu, can be used as biomarkers to further define processes which mediate propagation of biologic NPs and their contribution to disease.

Alkaline phosphatase (ALP) is an enzyme critical for physiological and pathological biomineralization. Experiments were designed to determine if ALP participates in formation of calcifying nanometer-sized particles (NPs) in vitro. Filtered homogenates of human calcified carotid artery, aorta and kidney stones were inoculated into cell culture medium containing 10% fetal bovine serum in the absence or presence of inhibitors of ALP or pyrophosphate. Calcific NP biofilm developed within one week after inoculation and their development was reduced by pyrophosphate and inhibitors of ALP. ALP protein and enzymatic activity were detected in washed NPs whether calcified or decalcified. Therefore, ALP activity is required for formation of calcifying NPs in vitro, as has previously been implicated during pathological calcification in vivo.

No abstract

No abstract

No abstract

No abstract

No abstract