Is there a pathological switch that triggers the onset of renal calcification?

IntroductionNephrocalcinosis, nephrolithiasis and Randalls plaque formation are distinct renal pathologies of biomineralization predominantly originating in the renal papillae. Experimental evidence on the events leading to the initial aggregation of nanometer-scale plaque or stone deposits in these regions are limited. Cellular plasticity is a regulatory mechanism of disease progression, and can lead to the transition of epithelial to mesenchymal stem-cell-like phenotypes, and generate macrophages to trigger pathophysiological alterations underlying renal biomineralization. We aim to understand the pathological mechanisms of biomineralization at the renal papillary tip of clinical patient samples and develop functional assays to analyze mechanisms of disease progression within organ-chip devices in vitro. MethodsWe analyzed clinical cohorts of patient renal papillae tissues obtained via nephrectomy (n=34) categorized as stone formers (SF) vs. non-stone formers (NSF). We studied the histopathology and genetic (bulk RNA-sequencing) composition of patient samples in the two groups. We examined the role of primary cells, including peripheral blood mononuclear cells (PBMCs) - progenitors of macrophages, isolated from patient blood samples to differentiate M1 pro and M2 anti-inflammatory macrophage phenotypes for static culture and flow/stretch analyses on organ-on-a-chip devices (Emulate Inc). We stained tissue sections with histology dyes and conducted digital pathology multiplexing analyses via quantitative pathology software (quPath, GitHub) by training an artificial neural network. We conducted fluorescence in situ hybridization (FISH) studies to identify genetic biomarkers of inflammation extracted from the bulk-RNA sequencing data. ResultsBased on the initial results of digital pathology, we identified renal calcium deposits (p value = 0.0017), collagen deposits (p value = 0.0001), fibrosis (p value = 0.0385) and renal casts or inflammatory cells among SF vs NSF cohorts across the cortex-to-tip region of renal papillae. Bulk RNA-sequencing analyses were primarily conducted with DAVID-KEGG and Panther 17.0 classification databases to highlight key regulatory pathways of interest involved at the onset of renal biomineralization, such as the oxidative stress pathway, hypoxia response via HIF activation, and inflammation mediated by chemokine and cytokine signaling. The FISH studies identified genes involved with inflammation; GALNT3, PLEKHO1, SLCO2A1, and VCAM1. We successfully differentiated patient-derived PBMCs to M1 and M2 macrophage lineages to study the impact of oxidative stress by using static 35 mm plate and flow microfluidic organ-chip instruments, to conduct appropriate functional assays in cell culture. ConclusionThe study outcomes provide insights to the precursors of renal biomineralization and delineated the expression of a pathological switch at the onset of hypoxia. The data will provide a fundamental framework to isolate primary cells from patient samples to conduct cell culture studies under static conditions, and translate the outcomes to flow analyses on a Kidney Chip instrument (Emulate. Inc) to mimic pathological conditions in a microphysiological environment in vitro. The ultimate outcome of this project will lead to the development of functional assays that emulate the kidney microphysiology on an organ-chip instrument, suited for clinical translation as a personalized, precision diagnostics and therapeutics platform.