Using samples from 101 volunteers and Western blot analysis of spot and 24 hr urine specimens, we have demonstrated that males who are confirmed active calcium oxalate (CaOx) stone formers (SF) typically expressed inter-alpha-trypsin inhibitor-trimer (IaTI-trimer) whereas age and ethnically matched normal (NRM) males expressed detectable levels of IaTI-trimer much less frequently (86 percent versus 23 percent; P is less than 0.0001). It is unlikely that the increased levels of IaTI- trimer in CaOx SF males are actively causing the episode of CaOx disease, because in females, who have a 2-3 fold lower incidence of CaOx disease than do males, the incidence of IaTI-trimer expression is similar in CaOx SF versus NRM females (94 percent versus 77 percent; P= 0.1938). IaTI-trimer has been shown to inhibit CaOx crystallization in vitro and has been recovered from the organic matrix of CaOx stones. It could be hypothesized that the increased expression of IaTI-trimer is in response to the damage caused by CaOx disease.
In Specific Aim 1, Western blot analysis will be utilized to determine if the expression of urinary IaTI-trimer is linked to active CaOx stone disease in males. In Experiment 1, the expression of urinary IaTI-trimer will be determined in urine samples from males: NRM, clinically active CaOx SF, and CaOx who have not had an episode in more than two years. Inactive statues will be confirmed by KUB. In Experiment 2, active male CaOx SF will be followed for 3 years from the time of clinical CaOx episode to determine if the incidence of urinary IaTi-trimer decreases during periods of inactive CaOx disease.
In Specific Aim 2, IaTI-trimer will be isolated from urine using gel and trypsin affinity chromatography. The isolated protein will be used as a standard to develop an ELISA to allow quantification of urinary IaTI-trimer.
In Specific Aim 3, the ELISA will be utilized to determine if IaTI-trimer is negatively regulated by androgens as the preliminary data suggests. Levels of urinary IaTI-trimer in males undergoing androgen deprivation for prostatic cancer, will be compared with those of age and ethnically matched NRM and CaOX SF. Lastly, a rat urolithiasis model (0.75 percent ethylene glycol and AIN-76 diet) will be utilized for formal hormonal deprivation/replacement studies. The rat model will also be utilized to determine if induction of urolithiasis increases the expression of IaTI- trimer systematically (i.e. the liver the primary source of serum IaTI- trimer), or locally (kidney) as judged by Northern blot analysis of RNA message for each of the IaTI-subunits and by Western blot/ELISA of the protein.
