Ausfällungsmechanismen von Ca-Oxalat in Gegenwart von Ca-Phosphaten und Osteopontin-Molekülen mit Bezug zur Bildung von Nierensteinen
Zusammenfassung der Projektergebnisse
Nephrolithiasis, the “kidney stone disease”, is a major health problem with increasing relevance. The majority of kidney stone consist of calcium phosphate (CaP) and calcium oxalate (CaOx). In these stones, the most common CaP phase is carbonated hydroxyapatite (HA) and CaOx mostly crystallizes as the monohydrate phase (COM; mineral name: whewellite). Both mineral phases may precipitate from urine within kidneys. However, in many cases CaOx precipitates at the surface of pre-existing CaP substrates. Such substrates are provided by CaP plaques (socalled Randall’s plaques), which form by calcification of connective tissue in the kidney and break through the epithelium at lesions, where the precipitated CaP comes in contact with oxalate-containing urine. So far two major hypotheses have been proposed: a) heterogeneous nucleation and growth of COM on CaP substrate from urine with metastable COM supersaturation; b) COM crystals form homogeneously in urine and aggregate at the surface of CaP plaques. In urine, crystal growth of COM is antagonized by inhibitor molecules, such as the protein osteopontin (OPN). The exact mechanism by which OPN inhibits COM crystal growth is currently investigated intensively in prospect of deriving a medical treatment for the prevention of CaOx kidney stones. This project aimed at a better understanding of CaOx kidney stone formation by following three interconnected paths: i) structural investigation of CaOx kidney stones with CaP plaques still attached; ii) experimental COM precipitation with OPN-derived peptides in the solution; iii) experimental precipitation of COM following our new hypothesis that CaP-COM mineral replacement reactions may play a role in CaOx kidney stone formation. i) Structural investigation of CaP-CaOx kidney stones revealed complex patterns of calcification with various structural characteristics within every single stone. Concerning the CaP plaques, we were surprised to find exceptionally well-preserved anatomical structures, such tubules of the nephrons and collagenous fibres of the connective tissue impregnated with CaP. Generally, CaP precipitated as particles <100 nm to 10 µm in size. Especially at the CaP-COM interface, a layer of small aggregated particles is found, probably corresponding to the epithelium. COM precipitated at the surface of the CaP plaque with various structures, massive stacked platelets, bundles of crystals and porous arrangement crystal platelets. These different structures suggest different environments and/or mechanisms of COM precipitation, which is supported by the finding of an organic layer intercalated at the CaP-COM interface in some places, while elsewhere the mineral phases are in immediate contact or there is no sharp interface at all. COM crystals show hierarchical structures essentially consisting of particles <100 µm. ii) Dissolved osteopontin-derived peptides of specific amino acid sequences show an inhibitory effect on COM crystal growth. The strongest effect was observed for a peptide especially rich in aspartate, while the effect of a phosphorylated peptide was significantly weaker. This finding suggests that negative charge density of the molecules is more important for inhibiting COM crystal growth than the degree of phosphorylation. As a result of the inhibition, crystal growth steps become scalloped and the crystal appears to consist of small particles corresponding to those observed in COM crystals of kidney stones. iii) Our hypothesis that mineral replacement may play a role in the formation of COM kidney stones attached to CaP plaque was experimentally validated with HA single crystals and bone immersed in urine simulating oxalate solutions. It turned out that nearly normal urine conditions can in fact induce COM precipitation on a CaP substrate by a coupled dissolution/precipitation reaction. In this process, only a fluid layer at the dissolving CaP surface becomes COM supersaturated by released Ca2+ which immediately re-precipitate with oxalate, while the bulk solution remains undersaturated. Similar observations were made using brushite crystals as a CaP substrate although mineral replacement was less effective due to the lower Ca2+ content of brushite. However, CaP-COM replacement could be an early stage of nephrolithiasis.
Projektbezogene Publikationen (Auswahl)
- Do mineral replacement reactions play a role in the formation of Ca-phosphate–Ca-oxalate kidney stones? Conference “Mechanisms of mineral replacement reactions”; final meeting of the “DELTA-MIN” Marie Curie Initial Training Network of the EU Seventh Framework Programme, Oviedo, Spain, 29.02.–02.03.2012
Sethmann, I., Kleebe, H.-J.
- Is mineral replacement a relevant mechanism in the formation of Ca-phosphate–Ca-oxalate kidney stones? Gordon Research Conference on Biomineralization, New London, New Hampshire, U.S.A., 12.–17.08.2012
Sethmann, I., Kleebe, H.-J.
- Mineral replacement mechanisms potentially involved in Ca-phosphate/Ca-oxalate kidney stone formation. 12th International Symposium on Biomineralization, Freiberg, Saxony, Germany, 27.–30.08.2013
Sethmann, I., Kleebe, H.-J., Grohe, B., Wendt-Nordahl, G., Knoll, T.
- (2014) Replacement of hydroxyapatite by whewellite: implications for kidney stone formation. Mineralogical Magazine 78(1), 105-114
Sethmann, I., Grohe, B., Kleebe, H.-J.
(Siehe online unter https://doi.org/10.1180/minmag.2014.078.1.07) - Ca-phosphate/Ca-oxalate mineral replacement potentially involved in kidney stone formation? The Granada-Münster Discussion Meeting on Crystal Growth and Biomineralization, Münster (Westfalen), Germany, 27.–28.11.2014
Sethmann, I., Kleebe, H.-J., Grohe, B., Wendt-Nordahl, G., Knoll, T.
