Abstract
Purpose: Identifying the source of stone formation in recurrent stone formers has always been a big problem. Material and Methods: In this study kidney stones from 52 patients were submitted to direct examination by scanning electronic microscopy (SEM) after manual fracture and 27 calculi were cultured in Eagle’s Minimum Essential Medium (E-MEM) and Brain-Heart Infusion (BHI) for eight weeks at 37°C in 5% CO2 atmosphere. Twenty-seven powdered and demineralized stones were suspended in sterile PBS, filtered through 0.22 m-pore-size sterile filters Millex (Millipore, Massachusetts, USA) and submitted to DNA extraction (Quiagen-Brazil). Platinum PCR SuperMIX (GIBCO-BRL), primers (Invitrogen), and Ultra Pure Water (Advanced Biotechnologies, Columbia, MD) were used for PCR (Polymerase Chain Reaction), which was generally conducted for 30 or 35 cycles with annealing of primers for 40 sec at 55°C and extension for 1 min at 72°C. Results: In 36 out 52 (69%) kidney stones it was detected the presence of biofilm coating the mineral surface of the stone when examined by SEM, consisting of coccoid particles, isolated or clustered, with diameter of 500 nm or less. Eleven out 27 (41%) kidney stone cultures produced white-colored sediment on the bottom of the tubes after eight-week incubation, revealing tiny structures similar to those observed directly by SEM. These structures were similar in size and morphology to spherical apatite particles previously observed in human kidney stones and named as nanobacteria (NB). No PCR products were observed in the samples. Conclusion: We found a strong correlation between renal stones and calcifying nanoparticles (CNP) in this study and these results open a new insight on this area to explore the etiology of stone formation. Whether NB/CNP are truly microorganisms or self-propagating mineral compounds is still controversial and its contribution, if any, in apatite nucleation and crystal growth remains uncertain.
Highlights
The prevalence of nephrolithiasis has been increasing in the last years, reflecting the gradual rise in living standards, affecting up to 5% of the general population in the USA
We found a strong correlation between renal stones and calcifying nanoparticles (CNP) in this study and these results open a new insight on this area to explore the etiology of stone formation
In this study we demonstrated the presence of a layer resembling biofilm on the mineral surface of 69% of the stones analyzed by scanning electronic microscopy (SEM), all showing coccoid nanoparticles bound together (Figure 1)
Summary
The prevalence of nephrolithiasis has been increasing in the last years, reflecting the gradual rise in living standards, affecting up to 5% of the general population in the USA. Stone formation in urinary tract is a multifactorial process that probably includes an imbalance between promoters of stone formation, such as urine supersaturation, crystal nucleation, aggregation and continued growth on the retained crystals, and their inhibitors, such as cit-. Rate and urinary glycoproteins [2]. Epidemiological studies have shown that most people affected are idiopathic stone formers and that only up to 20% of patients with renal calculi have predisposing factors [3]. It is well established that urease-producing gram-negative organisms are involved in the formation of some renal stones, mainly Proteus, but this occurs in less than 10% of patients [4]. NB are slow growing, not culturable in any standard microbiological
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