Nanobacteria-Induced Kidney Stone Formation: Novel Paradigm Based on the FERMIC Model Andrei P.
Sommer*,†
and E. Olavi
Kajander‡
Central Institute of Biomedical Engineering, University of Ulm, D-89081 Ulm, Germany, and Department of Biochemistry, University of Kuopio, 70211 Kuopio, Finland Received August 10, 2002;
CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 6 563-565
Revised Manuscript Received September 3, 2002
ABSTRACT: Mineralized nanobacteria have been found in a large number of human kidney stones. To analyze the mechanism of the stone formation induced by these crystalline biosystems, we demonstrate how free energy reduction by molecular interface crossing (FERMIC)sa powerful molecular transport mechanism derived from first principlesscould be implemented into existing concepts. FERMIC is generated by thermodynamic instabilities between nanoscopic molecular assemblies at proximal curvature asymmetries and predicts that kidney stones could be formed by material transfer between differently sized nanobacteria in interfacial proximity, immobilized for extended periods within the kidneys. Therapies counteracting kidney stone formation are suggested by the model and include biochemical and physical strategies allowing a reduction of the size differences as well as a minimization of the period of interfacial proximity between the nanobacteria passing the kidneys, principal factors recognized as essential for stone formation. Introduction There is ample evidence indicating that 90% of human kidney stones contain nanobacteria.1 The stone matrix consisted mainly of a conglomeration of nanobacteria and sedimentations of apatite, oxalate, struvite, and urate. Nanobacteria are assumed to play an essential role in the initiation and growth of kidney stones.2 They have a spheroid architecture with diameters from 80 to 300 nm in blood, reaching 10 µm in the kidney stones.1 Instead of a soft cell membrane, their outer surface consists of a nanocrystalline calcium phosphate shell (apatite) protecting a spherical cavity with a glutinous liquid in their interior.2 Nanobacteria have been observed to proliferate in cell culture, but it was not clear whether they could be considered as alive, in particular because of their extreme smallness and difficulty in isolating and analyzing their internal content.3 Some groups speculated that biomineralization attributed to nanobacteria could be initiated by nonliving macromolecules and self-propagating microcrystalline apatite.4 Recent observations could indicate, however, that the “lifeless picture” might be altered in favor of a novel paradigm: experiments showed uniform growth and replication of cultured nanobacteria under the impact of nonthermal light energy densities and intensities of the order of the solar constant.5,6 Degree and temporal evolution of the mineralization in liquid cell culture media seem to depend sensitively on two physical factors: interfacial space and relative size of the nanobacteria. Proximal particles with a disparity in size grow faster than populations with a narrow size distribution, clearly separated from one another. Distance and particle size-dependent growth phenomena are common in supersaturated solutions with crystallized components and in emulsions. Reduction of both free energy and concentration gradients is regarded as * To whom correspondence should be addressed. † University of Ulm. ‡ University of Kuopio.
the principal driving force for the diffusional material transfer in the coarsening phenomena called Ostwald ripening7 in supersaturated solutions containing differently sized, spatially separated crystalline particles8 and in ceramics.9 Alternatively, material transfer could be induced by local curvature asymmetries between differently sized nanoscopic objects in interfacial proximity, mediated by the free energy reduction by molecular interface crossing mechanism (FERMIC).10 Because there are no diffusional processes in FERMIC, the transport efficiency (material flux) is superior to Ostwald ripening. FERMIC has been verified to be valid for the description of various structure formation processes and transfer of molecules across differently curved proximal surfaces, in stationary biosystems,10 in thunderclouds with proximal ice crystal surfaces,11,12 and in noncontact imaging methods with the deposition of liquid material from the nanoscopic sensor tips onto the substrates examined (e.g., in near-field optical analysis).13,14 FERMIC-induced material transfer has been described in experiments employing perfluorocarbon (PFC)-based blood substitutes.10 FERMIC could be the dominant mechanism in biological processes such as bacterial conjugation, a process that promotes horizontal transfer of genetic material between donor and recipient cells in long-term physical proximity where both surfaces are coated with multispecies communities (biofilm), a phenomenon believed to be of evolutionary importance in bacteria15,16 and in nanobacteria.17 FERMIC has been proposed to be the dominant mechanism in the formation of giant PFC drops from clusters of nanoscopic droplets, trapped in liver and spleen, in an animal model. Laboratory experiments designed to simulate coarsening processes in theses storage organs showed that the giant drops found in these low mobility zones were not formed by coalescence, a spontaneous disappearance of the boundary layer between droplets in interfacial contact, inducing their subsequent fusion.10 In analogy to the interpretation and the analysis of the
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accumulation processes observed in liver and spleen, we propose that the majority of kidney stones nucleated by giant nanobacteria1,2 are primarily formed by the FERMIC mechanism. Review of the FERMIC Model For differently curved nanoscopic liquid droplets in a stationary emulsion10 and microscopic ice crystals with high curvature surfaces coated with liquid like layers and larger graupel particles in the turbulent environment of a thundercloud,11,12 it was possible to derive a powerful mechanism pumping molecules from high curvature surfaces to proximal low curvature surfaces solely from first principles. From the van der Waals model describing the intermolecular interaction between adjacent curved surfaces, the maximum molecular instability (fluctuations, most probable transfer of molecules) is expected to occur in the near-field of the area around the point of contact. In the simplest formulation of the model, surface molecules are attracted anisotropically by a lesser number of closest neighbors than in the bulk liquid. Realizing that the coordination number of surface molecules significantly decreases with increasing surface curvature, the model could be generalized for unequally curved surfaces. In a two-dimensional (2D) analogue of an idealized three-dimensional model formulated for two spherical objects of radius R1, R2, it had been shown that for two molecules belonging to two proximal surfaces, the difference of the interaction energies, ∆Eos ) Eso1 - Eso2, is given by ∆Eos ) (1/4π)zs ‚ rw ‚ WAA[(1/R2) - (1/R1)], where zs denotes the coordination number of a molecule at a plane surface, rw is the range of the attractive intermolecular forces centered around a surface molecule, and WAA represents the pair interaction energy. This equation, rigorously derived for a 2D configuration and experimentally verified for nanoscale objects in interfacial proximity,10-12 allows the determination of the sign of the net interaction energy ∆Eos. By noting that the pair interaction energy WAA is a negative energy, it is clear from the above equation that ∆Eos becomes negative for R1 > R2. Following the relation defining the quantity ∆Eos, a negative ∆Eos is equivalent with Eso2 > Eso1. Thus, a molecule at the side of the smaller object should be less stable, having a higher probability for fluctuations than corresponding molecules at the surface of the larger object. For two nanobacteria of radius R1, R2 at a relative distance rw , (R1, R2), FERMIC predicts a transfer of the solvated calcium phosphate components and/or internal liquid content17 from the smaller to the bigger particle. This is because the system tends to reduce its potential energy by an evolution toward the maximal thermodynamic stability. Material transport induced by FERMIC has the same direction as characteristic for Ostwald ripening, operating in stationary systems at large distances in the presence of spatial concentration gradients, driving molecules from the smaller to bigger particles. Under spatially restricted stationary conditions, a synergistic and complementary interplay of the two mechanisms seems possible. Interestingly, solitary nanobacteria and clusters of stressexposed nanobacteria create around their apatite shells a mucus, low mobility environment. The presence of mucus is associated with two constrains: temporally
Sommer and Kajander
extended interfacial proximity and restricted spatial migration of dissolved apatite components, potentially securing nanobacterial survival by accelerating their growth and inducing rapid kidney stone formation.2 Remarkably, the secreted mucus also has a strong anticlotting effect on blood.5 From their abundance in the kidney stones and blood tests in healthy persons, we concluded that nanobacteria could be more common in the human body than previously assumed.2 Responding to environmental stress by releasing large amounts of growth-stimulating mucus, they could thus contribute to the retarded healing of wounds experienced by astronauts exposed to microgravity for prolonged periodssa physiological state characterized by abnormal blood circulation with accumulations of blood in the upper parts of the body. Discussion The FERMIC model reviewed here could be regarded as a primitive approximation allowing a better understanding of the physicochemical mechanisms potentially facilitating the transfer and the incorporation of molecules or parts of macromolecular cluster associations into nanobacteria, which are supposed to occur during their accretion in the kidneys. This understanding could be beneficial to analyze and possibly to influence biomineralization and growth processes locally. Experimental evidence for a multifold increase in the size of nanobacteria isolated from the kidneys, as compared to the mean particle size found in blood, strongly supports the proposed paradigm. FERMIC processes have been primarily studied in emulsions, in low mobility environments, e.g., in organs such as liver and spleen, or between ice crystals and graupel in thunderclouds. Most of the interfacial transfer processes involving nanobacteria are expected to occur in the low mobility environment of the kidneys (papillary area and large collecting ducts) with elevated nanobacterial concentrations, a situation where two differently curved surfaces are separated by a liquid layer creating loose adhesion and nanobacterial colony formations.2 The presence of the liquid layer separating proximal nanobacteria interacting prior to molecular recognition via eventual surface receptor contacts could be crucial for a material transport induced by geometrical asymmetry, a process similar to the transfer of water molecules across an interfacial barrier of compressed air separating two colliding hydrometeors of different radii of curvature.11 Similarly, the mucus layer could modulate the directional transfer of molecules across the interface between the nanobateria, practically increasing preexistent size imbalances. Conclusions Depending on differences in polarity and the interfacial proximity determined by the thickness of the liquid layer separating nanobacteria from each others parameters that could be calculated via computer simulationsit should be possible to control the relative interaction energy between mobile nanobacteria and immobilized nanobacterial colony plaques, by minimizing or inhibiting the release of mucus promoting adhesion and material transfer between proximal apatite
Nanobacteria-Induced Kidney Stone Formation
surfaces. Presumably, this could be achieved by the use of biochemical methods. Indeed, the administration of antimicrobial agents has been shown to inhibit nanobacterial replication, limiting their growth irreversibly.17 In addition, nanobacteria treated with polarized white light showed a pronounced tendency to keep equiradical distributions and a reduced level of surrounding mucus.5 Hence, both routes, the physical and the chemical, and most likely their combination, seem now viable in attacking the stone problem. Additional research on this multidisciplinary field is a significant challenge to scientists trying to unravel the complexity of molecular transport phenomena and modeling crystallization processes in biosystemssa multifaceted field that could provide novel noninvasive and preventive therapies. References (1) Ciftcioglu, N.; Bjo¨rklund, M.; Kuorikoski, K.; Bergstro¨m, K.; Kajander, E. O. Kidney Int. 1999, 56, 1893. (2) Kajander, E. O.; Ciftcioglu, N.; Miller-Hjelle, M. A.; Hjelle, J. T. Curr. Opin. Nephrol. Hypertens. 2001, 10, 445.
Crystal Growth & Design, Vol. 2, No. 6, 2002 565 (3) Abbott, A. Nature 2000, 408, 394. (4) Cisar, J. O.; Xu, D. Q.; Thompson, J.; Swaim, W.; Hu, L.; Kopecko, D. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11511. (5) Sommer, A. P.; Hassinen, H. I.; Kajander, E. O. J. Clin. Laser Med. Surg. 2002, 20, 241. (6) Sommer, A. P.; Oron, U.; Kajander, E. O.; Mester, A. R. J. Proteome Res. 2002, ASAP article. (7) Ostwald, W. Z. Phys. Chem. 1900, 34, 495. (8) Wagner, C. Z. Elektrochem. 1961, 65, 581. (9) Shen, Z.; Zhao, Z.; Peng, H.; Nygren, M. Nature 2002, 417, 266. (10) Sommer, A. P.; Ro¨hlke, W.; Franke, R. P. Naturwissenschaften 1999, 86, 335. (11) Sommer, A. P.; Levin, Z. Atmos. Res. 2001, 58, 129. (12) Sommer, A. P. Langmuir 2002, 18, 5040. (13) Sommer, A. P.; Franke, R. P. Micron 2002, 33, 227. (14) Sommer, A. P.; Franke, R. P. J. Proteome Res. 2002, 1, 111. (15) Ochman, H.; Lawrence, J. G.; Groisman, E. A. Nature 2000, 405, 299. (16) Ghigo, J. M. Nature 2001, 412, 442. (17) Ciftcioglu, N.; Miller-Hjelle, M. A.; Hjelle, J. T.; Kajander, E. O. Antimicrob. Agents Chemother. 2002, 46, 2077.
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