Presentation Authors: Mohammad Makki, Seth Winfree, Indianapolis, IN, Elaine Worcester, CHicago, IL, Frank Witzmann, James Lingeman, James Williams, Tarek El-Achkar*, Indianapolis, IN
Introduction: The pathogenesis of kidney stones in humans is poorly understood. There are multiple types of kidney stones, and the role of inflammation in promoting specific stone types is not well established. Here, we applied a combination of unbiased omics and quantitative large-scale 3D imaging on kidney stones and papillae from two types of stone forming patients: calcium oxalate and brushite. Our goal was to uncover a specific inflammatory signature that could differentially associate with a specific type of stone disease and infer how they are formed.
Methods: Kidney stones were obtained from a well-characterized cohort of patients in a high volume clinic. Stones were further processed for label free quantitative mass spectrometry to quantify differentially expressed proteins. Papillary biopsies were obtained at the time of percutaneous nephrolithotomy and processed for histology and large scale 3D imaging, as well as transcriptomics. 3D tissue cytometry and analysis was done using Volumetric Tissue Cytometry and Analysis Software (VTEA).
Results: Brushite stones have differentially increased neutrophil proteins such as myeloperoxidase and elastase compared to calcium oxalate evaluated by LC/MS. To determine if this observation is driven by changes in the kidney papillae, we confirmed that the number (6.91 Â±1.19% vs. 1.35 Â± 0.28%; p=3.2x10-5) and the density of neutrophils (10.1x103 Â±6.2x103/mm3 vs. 0.91x103 Â±0.67x103/mm3; p < 0.05) were increased in the papillae from brushite vs. calcium oxalate patients, respectively. To explain how neutrophil proteins are transitioning from the tissue to the stone matrix, we investigated whether Neutrophil Extracellular Trap (NET) formation, whereby neutrophils expel their DNA and their cytoplasmic content, is more common in brushite stones. Indeed, histone citrullination of neutrophils, a marker of NETosis, was increased in brushite compared the calcium oxalate papillae (1.3 Â± 0.28% vs. 0.07 Â±0.04%; p < 0.05). Biopsy total RNA also showed increases in brushite for inflammation and neutrophil activation pathways.
Conclusions: Our work supports that increased neutrophil infiltration and NETosis may be an important factor in the pathogenesis of brushite stones. We propose that metabolic changes or ascending infections could trigger a hyper-inflammatory response that leads to the release of NET proteins in the urine, which promote brushite stone formation.
Source of Funding: NIH P01 DK056788