Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers

Abstract

Two proteins of 17 and 24 kDa, respectively, which were immunologically related to bikunin, were purified from urine of healthy men, using in the last step a trypsin CNBr-sepharose affinity column. These proteins strongly inhibited calcium oxalate (CaOx) crystallization in two in vitro models. In the first model, the presence of 8 microg/ml protein in a medium containing 0.76 mM CaCl2 (with 45Ca) and 0.76 mM ammonium oxalate inhibited the crystallization process by 80%, as estimated by supernatant radioactivity after 60 min of incubation. A similar inhibition was observed in the second turbidimetric model, where the CaOx crystallization kinetics were followed for 10 min at 620 nm in a medium containing 4 mM CaCl2 and 0.5 mM Na2Ox. These proteins were used as standard protein for the development of an enzyme-linked immunosorbent assay (ELISA) in urine. Mean (+/-SEM) urinary bikunin concentration in 18 healthy subjects was 5.01 +/- 0.91 microg/ml. This was a concentration range of strong inhibitory activity in vitro. Bikunin values were nearly 50% lower (2.54 +/- 0.42 microg/ml, P=0.007) in 31 CaOx renal stone formers (having weddelite crystals in their first morning urine) than in the healthy volunteers. A correlation was found between urinary bikunin and alpha-1 microglobulin concentrations in the control group (y=0.73x + 1.09, r2=0.8) while no such correlation existed in the lithiasis group. In conclusion, bikunin exerts a strong inhibitory action of CaOx crystallization in vitro. Its involvement in urinary CaOx crystallization of stone formers is highly probable, based on the significant decrease in its urinary concentration in the majority of stone formers studied.

Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers

“…Medetognon-Benissan et al (27) reported that BK levels were lower in SF than in healthy controls. By contrast, Suzuki et al (43) found higher urinary concentrations of BK in male SF compared with healthy male controls and that a greater proportion of SF than normal patients had an aberrant BK 25-kDa species, which is identified to be deglycosylated BK, a form with reduced effectiveness in inhibiting CaOx crystal growth.…”

“…Calgranulin is a strong inhibitor of CaOx crystal growth and consists of two monomeric subunits, A and B (also known as MRP8 and MRP14, or S100A8 and S100A9), which are small (11 and 14 kDa, respectively) acidic proteins that can combine to form homo-and heterodimers (24)(25)(26)(27)(28), as well as tetramers and other higher-order multimers (35-48 kDa) (16,31,34,44). In fact, it is the calA/calB heterodimer for which crystal growth inhibition has been documented (34).…”

“…Such proteins include TammHorsfall protein (THP) (20), osteopontin (uropontin) (2), prothrombin fragment 1 (PF1) (38), inter-␣-trypsin inhibitor (ITI) (9), and bikunin (BK), a portion of ITI (3), calgranulin (34), CD59 (7), and albumin (10). Although some reports suggest defects, deficiencies, or variations of these molecules among SF vs. normal subjects (13,14,26,27,43), it remains unclear whether any of these is a cause or a consequence of stones; no research thus far has translated molecular knowledge into clinical practice.…”

Effects of inter-α-inhibitor and several of its derivatives on calcium oxalate crystallization in vitro

“…There are several published estimates of the concentration of bikunin in human urine : 500 nmol\l [47] ; 10.13p1.13 µg\ml (280.5 nmol\l) and 6.72p0.93 µg\ml (186 nmol\l) in normal men and women respectively [49] ; 5.01p0.91 µg\ml (208 nmol\l) [50] ; and 4.82p 2.46 mg\day (138 nmol\l) and 3.86p1.35 mg\day (110 nmol\l) in normal men and women respectively [51]. The lowest estimate of urinary bikunin to date is 0.25p0.11 µg\mg of creatinine [52].…”

Comparison of Fibronectin Content in Urinary Macromolecules between Normal Subjects and Recurrent Stone Formers

“…Benissan et al [12]reported that bikunin, described as a potent inhibitor of CaOx crystallization in vitro, was lower in urine from CaOx renal stone formers than healthy volunteers. Yasui et al [13]demonstrated that the excretion of urinary OPN in stone formers might be lowr than in controls with no history of urolithiasis.…”