Multicolor imaging of calcium-binding proteins in human kidney stones for elucidating the effects of proteins on crystal growth

Abstract

The pathogenesis of kidney stone formation includes multi-step processes involving complex interactions between mineral components and protein matrix. Calcium-binding proteins in kidney stones have great influences on the stone formation. The spatial distributions of these proteins in kidney stones are essential for evaluating the in vivo effects of proteins on the stone formation, although the actual distribution of these proteins is still unclear. We reveal micro-scale distributions of three different proteins, namely osteopontin (OPN), renal prothrombin fragment 1 (RPTF-1), and calgranulin A (Cal-A), in human kidney stones retaining original mineral phases and textures: calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD). OPN and RPTF-1 were distributed inside of both COM and COD crystals, whereas Cal-A was distributed outside of crystals. OPN and RPTF-1 showed homogeneous distributions in COM crystals with mosaic texture, and periodically distributions parallel to specific crystal faces in COD crystals. The unique distributions of these proteins enable us to interpret the different in vivo effects of each protein on CaOx crystal growth based on their physico-chemical properties and the complex physical environment changes of each protein. This method will further allow us to elucidate in vivo effects of different proteins on kidney stone formation.

Roles of heat-shock protein 90 and its four domains (N, LR, M and C) in calcium oxalate stone-forming processes

“…After the COM crystals form, they can grow continuously under the supersaturated condition by adding free Ca 2+ and C 2 O 4 2− ions to the crystals [ 61 ]. Immunofluorescence staining has revealed the spatial distribution of negatively charged proteins inside and outside of the crystals during growing process [ 19 ]. In our present study, negatively charged FL-HSP90, N-HSP90 and C-HSP90, and positively charged M-HSP90 could enhance the growth of COM crystals.…”

“…Additionally, alterations of protein expression in renal tubular epithelial cells (RTECs) by COM crystals have been identified via proteomics approach [ 8 – 17 ]. Interestingly, functional analyses have demonstrated that some of such altered proteins play roles as modulators (promoters or inhibitors) [ 18 , 19 ] and COM crystal receptors [ 20 – 22 ] during the stone-forming processes.…”

Quantitative analysis of calcium oxalate monohydrate and dihydrate for elucidating the formation mechanism of calcium oxalate kidney stones

“…The final structure of kidney stones and/or phase transformation speed estimated from the structure suggests the history of kidney stone growth and urine environment changes. Combined with our recent technique, multicolor imaging of calcium-binding proteins in human kidney stones [9], we can evaluate various kidney stone samples, and the information that stones have will reveal the factors of the stone formation processes and the interaction with organic molecules.…”

“…The polymorphism of CaOx in vivo is crucial for understanding the stone formation process. Further investigation using quantitative measurement procedures of CaOx combined with the visualization of multiple proteins in human kidney stones [9] will elucidate the kidney stone formation processes.…”

“…Recently, the observation and analysis of kidney stone thin sections were focused on again, and we developed a new method for making thin sections without severe deterioration of the surface and protein expression. Using such thin sections of kidney stone, we succeeded to reveal micro-scale distributions of three different proteins using the advanced multiple immunofluorescence staining (multi-IF staining) [9]. The spatial distributions of these proteins in kidney stones are essential for evaluating the in vivo effects of proteins on stone formation.…”