Collagen fibrils and cell nuclei are entrapped within Randall's plaques but not in CaOx matrix overgrowth: A microscopic inquiry into Randall's plaque stone pathogenesis

Abstract

Calcium oxalate (CaOx) stones can grow attached to the renal papillary calcification known as Randall's plaque. Although stone growth on Randall's plaque is a common phenomenon, this mechanism of stone formation is still poorly understood. The objective of this study was to investigate the microenvironment of mature Randall's plaque, explore its molecular composition and differentiate plaque from CaOx overgrowth using multimodal imaging on demineralized stone sections. Fluorescence imaging showed consistent differences in autofluorescence patterns between Randall's plaque and calcium oxalate overgrowth regions. Second harmonic generation imaging established the presence of collagen only in regions of decalcified Randall's plaque but not in regions of CaOx overgrowth matrix. Surprisingly, in these stone sections we observed cell nuclei with preserved morphology within regions of mature Randall's plaque. These conserved cells had variable expression of vimentin and CD45. The presence of nuclei in mature plaque indicates that mineralization is not necessarily associated with cell death. The markers identified suggest that some of the entrapped cells may be undergoing dedifferentiation or could emanate from a mesenchymal or immune origin. We propose that entrapped cells may play an important role in the growth and maintenance of Randall's plaque. Further characterization of these cells and thorough analyses of the mineralized stone forming renal papilla will be fundamental in understanding the pathogenesis of Randall's plaque and CaOx stone formation.

Keywords: Randall's plaque; calcium phosphate; cells; kidney; kidney stones; nuclei; renal calculi; renal papilla; stones; urinary calculi/etiology.

Figure 1

Demineralization of a Randall’s Plaque (RP) stone. A: A 3-D reconstruction of calcium oxalate (CaOx) stone surface growing on RP using micro-CT before the demineralization treatment. B: The same RP stone from 1A suspended in gel and completely demineralized. Note that the structure of the stone is completely retained, even though no mineral was detectable by micro-CT. C: This section, in maximum intensity z-projection, shows RP in blue (arrowhead) and the CaOx matrix in the green-yellow range. An artifactual void between the outer edge of the stone and the edge of the CaOx overgrowth can be seen (white arrows) indicating that shrinkage of CaOx matrix away from original boundary has occurred (probably during the dehydration process before paraffin embedment). Scale bar = 250 μm.

Figure 2

Second harmonic generation (SHG) of decalcified Randall’s plaque shows collagen fibrils. A: Autofluorescence in region of demineralized Randall’s plaque (stone different from the one in in Figure 1). Spaces of apparent cell nuclei in plaque are seen. B: Second harmonic generation (SHG) visualization of collagen fibrils (arrowheads) in specimen shown in A. SHG shows collagen fibers in part of Randall’s plaque but not throughout the entire plaque region. Region of plaque devoid of SHG signal is indicated by an asterisk. C: Randall’s plaque stone from patient 2 (table 1), histologically sectioned through a different plane, showing a plaque region (in blue) and the surrounding CaOx matrix. D: SHG of section C shows that collagen fibrils (arrowheads) were observed coursing throughout the plaque section (scale bars = 25 μm).

Figure 3

Cell nuclei in demineralized Randall’s plaque stain with 7-AAD or DAPI. A: A specific region of a demineralized Randall’s plaque stone (white arrowhead) (from patient 1) shows enriched cell nuclei positivity with 7-AAD (white arrowhead, nucleus). B: A Randall’s plaque stone section from patient 2 displaying a few cell nuclei at the edge of the demineralized plaque, presumably from the tissue side. Notice the quantitative differences between the numerous nuclei in A and sparser nuclear region in B (scale bar = 25 μm). C: Enlarged region of cell nuclei in plaque stained with DAPI from patient 4. This stone section was quenched with 50mM NH4Cl to reduce native fluorescence from the stone (scale bar = 5 μm).

Figure 4

Entrapped cell nuclei on Randall’s plaque stones express vimentin or CD45. A: Randall’s plaque stone section showing nuclei positivity with both DAPI and VIM (white arrowheads) (scale bar = 100 μm). B: Inlet of A (scale bar = 5 μm). C: A cortical section of human kidney showing DAPI (blue) and VIM positivity (red) (scale bar = 5 μm). D: Randall’s plaque stone with 7-AAD (red) positive nuclei with variable CD45 (green) positive expression (scale bar = 25 μm). E: Inlet of D (scale bar = 5 μm). F: A cortical section of human kidney showing 7-AAD (red) positive nuclei (white arrowhead) and CD45 expression (green).