Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men.
OBJECTIVE
We determine if the immunoreactive profile of urinary inter-alpha-trypsin inhibitor can be used to distinguish between normal individuals and individuals with calcium oxalate stone disease.


METHODS
Urinary proteins were dialyzed against water (15 kDa. molecular weight cutoff), lyophilized and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% acrylamide, reducing conditions) followed by Western blot. Inter-alpha-trypsin immunoreactive proteins were detected by enhanced chemiluminescence. Stone formation was confirmed to be active radiologically or passed as stone or gravel within 12 months of the sample. Stone composition was confirmed crystallographically. Normal individuals had no personal or familial history of urolithiasis and matched stone forming patients regarding race (white) and age (23 to 71 years old). Urine from a total of 101 individuals was analyzed.


RESULTS
The intact inter-alpha-trypsin trimer (approximately 220 to 240 kDa.) and heavy chain (HC) 2-bikunin/HC1-bikunin dimers (approximately 115 to 130 kDa.) were detected more often in stone forming men (23 of 26 [89%] and 26 of 26 [100%], respectively) than in normal individuals (6 of 26 [23%] and 5 of 26 [19%], respectively, p < 0.0001). In those normal individuals who expressed inter-alpha-trypsin trimer and HC-bikunins the relative intensities were 5.3+/-1.4% and 16.3+/-17.1% of the stone forming controls, respectively. The identity of high molecular weight-inter-alpha-trypsin immunoreactive bands was confirmed using antibodies against the individual subunits (HC1, HC2, HC3, bikunin). In contrast to men high molecular weight-inter-alpha-trypsin's were readily detected in normal and stone forming women with equal frequency (inter-alpha-trypsin-trimer p=0.1337, HC-bikunins p=0.2836): inter-alpha-trypsin-trimer 17 of 18 [94%] and 9 of 13 [77%]; HC-bikunins 17 of 18 [94%] and 10 of 13 [85%]). Inter-alpha-trypsin-trimer and HC-bikunins, respectively, were detected in 2 and 5 of 10 patients with chronic renal disease. Expression was not related to hematuria or proteinuria.


CONCLUSIONS
Immunoreactive profiles of urinary proteins may be able to be developed into a useful diagnostic tool to identify active stone formation, although a separate panel may be required for men and women. It is possible that these differences may provide clues as to why the incidence of stone disease is higher in men than women.
Marengo
S R
SR
James and Eilleen Dicke Research Laboratory, Department of Urology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Resnick
M I
MI
Yang
L
L
Chung
J Y
JY
D000328:Adult; D002129:Calcium Oxalate / Q000652:urine*; D005260:Female; D006023:Glycoproteins; D006801:Humans; D007150:Immunohistochemistry; D007669:Kidney Calculi / Q000737:chemistry* / Q000652:urine*; D008297:Male; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D015842:Serine Proteinase Inhibitors / Q000652:urine*; D012737:Sex Factors; D014360:Trypsin Inhibitor, Kunitz Soybean; D000515:alpha 1-Antitrypsin / Q000652:urine*
D006023:Glycoproteins; D008562:Membrane Glycoproteins; C109148:SPINT2 protein, human; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D002129:Calcium Oxalate; D014360:Trypsin Inhibitor, Kunitz Soybean