Crystalluric and tubular epithelial parameters during the onset of intratubular nephrocalcinosis: illustration of the 'fixed particle' theory in vivo

Background. The fixed particle theory states that, besides crystal formation in the tubular fluid, crystal adhesion to the tubular epithelium is a prerequisite for the development of intratubular nephrocalcinosis. It has been hypothesized that the tubular epithelium, in order to bind crystals, needs to be phenotypically altered. Whereas most evidence hereto is provided by in vitro experiments, we set out to illustrate this theory in vivo. Methods. We simultaneously investigated the temporal changes of nephrocalcinosis-associated parameters during and shortly after a 4-day ethylene glycol (EG)-administration period in rats. We measured oxaluria, crystal formation, crystalluria, apoptosis, epithelial injury/ regeneration and luminal membrane expression of several crystal-binding molecules [hyaluronan (HA), osteopontin (OPN) and for the first time in vivo, annexin-2 (ANX2) and nucleolin-related-protein (NRP) and one of their receptors (CD44, HA/OPN-receptor]. Clinically, renal biopsies of preterm infants, transplant patients and acute phosphate nephropathy patients were stained for ANX2, NRP, HA and OPN. Results. In the presence of a rather constant and persistent intratubular crystal formation, crystal retention gradually increased during EG-administration and markedly increased after arrest thereof, indicating that the development of crystal adhesion requires more than just the presence of crystals in the tubular fluid. All luminal membrane markers and a regenerating/dedifferentiated epithelium, unlike apoptosis, to various extents were upregulated concurrently and in association with crystal adhesion. However, both in humans and rats, expression of luminal molecules was not confined to crystal-containing tubules. Conclusions. Altogether, these findings allow better insight into the mechanisms underlying the fixed particle theory in vivo and indicate that an altered epithelial phenotype with crystal-binding properties precedes crystal adhesion, thereby corroborating the requirement of tubular epithelial phenotypical changes in the development of intratubular nephrocalcinosis.