title: Citrate determines calcium oxalate crystallization kinetics and crystal morphology-studies in the presence of Tamm-Horsfall protein of a healthy subject and a severely recurrent calcium stone former.
abstract: BACKGROUND
  The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm-Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate.
  
  
  METHODS
  Nucleation and aggregation of CaOx crystals from a supersaturated, stirred solution (200 mM NaCl, 10 mM Na-acetate, pH 5.70, 5 mM Ca and 0.5 mM Ox) were studied by spectrophotometric time-course measurements of OD at 620 nm (OD(620)). Measured parameters were induction time t(I) (time to induce formation of detectable particles), S(N), (slope of increase of OD(620), mainly due to crystal nucleation), and S(A), (slope of decrease of OD(620) after equilibrium has been reached, due to crystal aggregation). Effects of citrate, nTHP and SF-THP on these parameters were measured, and scanning electron microscopy (SEM) was performed.
  
  
  RESULTS
  At 1.5, 2.5 and 3.5 mM, citrate increased t(I) and inhibited crystal nucleation (by 78-87%) as well as aggregation (by 63-70%), and smaller CaOx crystals (length/width ratio 1.7+/-0.1) than under standard conditions (length/width 3.9+/-0.5) were visible (P<0.001). Normal THP at 30 and 40 mg/l inhibited crystal nucleation and, more strongly, aggregation (inhibition 76-81%). SEM revealed a decrease in length/width ratio to 2.6+/-0.4 (P=0.051 vs standard conditions) and less aggregation than without nTHP. At all concentrations tested, SF-THP reduced t(I) (P=0.0001 vs standard conditions) and promoted aggregation (inhibition -48 to -33%); crystals were elongated with a length/width ratio of 4.3+/-0.6 (P<0. 05 vs nTHP). When simultaneously present with nTHP, citrate enhanced the inhibitory effects of nTHP, producing the smallest (length/width 1.5+/-0.1) and least aggregated crystals. Finally, 3.5 mM citrate turned promotory SF-THP into a crystallization inhibitor with abundant small and clustered, but not aggregated crystals.
  
  
  CONCLUSIONS
  Citrate appears to be the main determinant of CaOx crystallization rates and crystal morphology in the presence of nTHP as well as SF-THP. Its effects appear to predominate over those of THP, since even promotory SF-THP is turned into a crystallization inhibitor in the presence of citrate. This re-emphasizes at a morphological level what has been concluded from functional as well from clinical studies, namely that citrate is needed in urine at equimolar concentrations to calcium in order to prevent the formation of large crystal aggregates in presence of abnormal THP.
authors.affiliation: Department of Medicine and Laboratory of Electron Microscopy, University of Berne, Berne, Switzerland.
authors.forename: B
authors.initials: B
authors.lastname: Hess
authors.forename: S
authors.initials: S
authors.lastname: Jordi
authors.forename: L
authors.initials: L
authors.lastname: Zipperle
authors.forename: E
authors.initials: E
authors.lastname: Ettinger
authors.forename: R
authors.initials: R
authors.lastname: Giovanoli
mesh_terms: D000328:Adult; D002129:Calcium Oxalate / Q000737:chemistry*; D019343:Citric Acid / Q000494:pharmacology*; D003460:Crystallization; D003461:Crystallography; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007669:Kidney Calculi / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D009088:Mucoproteins / Q000494:pharmacology*; D012016:Reference Values; D058949:Uromodulin
chemical_list: D009088:Mucoproteins; C548612:UMOD protein, human; D058949:Uromodulin; D002129:Calcium Oxalate; D019343:Citric Acid