Wilson, Elena

Background and objectives Urinary stone disease has been associated with inflammation, but the specific cell interactions that mediate events remain poorly defined. This study compared calcification and inflammatory cell patterns in kidney tissue from radical nephrectomy specimens of patients without and with a history of urinary stone disease. Design, setting, participants, & measurements Nontumor parenchyma of biobanked radical nephrectomy specimens from age- and sex-matched stone formers ( n =44) and nonstone formers ( n =82) were compared. Calcification was detected by Yasue staining and inflammatory cell populations by immunohistochemistry for CD68 (proinflammatory M1 macrophages), CD163 and CD206 (anti-inflammatory M2 macrophages), CD3 (T lymphocytes), and tryptase (mast cells). Calcifications and inflammatory cells were quantified in cortex and medulla using Image-Pro analysis software. Results Calcification in the medulla of stone formers was higher than in nonstone formers ( P <0.001). M1 macrophages in the cortex and medulla of stone formers were greater than in nonstone formers ( P <0.001), and greater in stone former medulla than stone former cortex ( P =0.02). There were no differences in age, sex, body mass index, tumor characteristics (size, stage, or thrombus), vascular disease status, or eGFR between the groups. M2 macrophages, T lymphocytes, and mast cells did not differ by stone former status. There was a correlation between M1 macrophages and calcification in the medulla of stone formers (rho=0.48; P =0.001) and between M2 macrophages and calcification in the medulla of nonstone formers (rho=0.35; P =0.001). T lymphocytes were correlated with calcification in the cortex of both nonstone formers (rho=0.27; P =0.01) and stone formers (rho=0.42; P =0.004), whereas mast cells and calcification were correlated only in the cortex of stone formers (rho=0.35; P =0.02). Conclusions Higher medullary calcification stimulated accumulation of proinflammatory rather than anti-inflammatory macrophages in stone formers.

Background Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1–V3, V3–V5) and internal transcribed spacer (ITS1, ITS2) regions. Results Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes , Proteobacteria , and Aspergillus niger . Bacterial cells approximately 1 μm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.