Volume 53, Issue 6, Pages 1727-1735 (June 1998) Identification of structural domains in inter-α-trypsin inhibitor involved in calcium oxalate crystallization  Hiroshi Kobayashi, Kiyoshi Shibata, Michio Fujie, Dan Sugino, Toshihiko Terao  Kidney International  Volume 53, Issue 6, Pages 1727-1735 (June 1998) DOI: 10.1046/j.1523-1755.1998.00940.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Schematic representation of IαI and its derivatives. IαI protein is composed of two heavy chains (HC1 and HC2) and one light chain associated with chondroitin sulfate. The light chain, also known as bikunin or urinary trypsin inhibitor (UTI), has two active Kunitz-type protease inhibitor domains and the inhibitory activity of the IαI protein entirely depends on UTI. Schematic representation of the biosynthesis of IαI and derivatives was described29,38,39. Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Calcium oxalate (CaOx) crystallization inhibitory activity of IαI, heavy chains of IαI, UTI, chondroitinase-treated UTI, and HI-8. CaOx monohydrate crystal growth inhibition assay with a tracer of [14C]oxalate: Calcium and oxalate were mixed with a trace amount of [14C]oxalate in the presence of IαI and its derivatives (0 to 10-5 M). Symbols are: (•) IαI; (□) heavy chains of IαI; (○) UTI; (▪) chondroitinase AC-treated UTI; (▵) HI-8. Results are the mean of two experiments, carried out on triplicate assays. Data are mean ± SD. Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Seed crystals after incubation with and without IαI or its derivatives. Light micrographs of crystal deposited. (A) Typical CaOx crystals. The crystals were precipitated from seeded CaOx crystals supplemented with 1 μM IαI (B), heavy chains of IαI (C), UTI (D), chondroitinase AC-treated UTI (E), and HI-8 (F). Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Seed crystals after incubation with and without IαI or its derivatives. Light micrographs of crystal deposited. (A) Typical CaOx crystals. The crystals were precipitated from seeded CaOx crystals supplemented with 1 μM IαI (B), heavy chains of IαI (C), UTI (D), chondroitinase AC-treated UTI (E), and HI-8 (F). Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Seed crystals after incubation with and without IαI or its derivatives. Light micrographs of crystal deposited. (A) Typical CaOx crystals. The crystals were precipitated from seeded CaOx crystals supplemented with 1 μM IαI (B), heavy chains of IαI (C), UTI (D), chondroitinase AC-treated UTI (E), and HI-8 (F). Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IαI or its derivatives on crystal growth and aggregation in a seeded metastable solution of CaOx. (A) Effect of UTI on CaOx crystal growth, expressed as the increase in crystal diameter by flow cytometry. Panel 1, UTI concentration = 0 μM and CaOx crystal diameter = 40.6 μm; panel 2, 0.2 μM and 15.3 μm; panel 3, 1.0 μM and 2.40 μm; panel 4, 5.0 μM and 2.01 μm; panel 5, 25 μM and 1.64 μm; panel 6, 100 μM and 1.93 μm. Particle diameter standards are shown on the far right. Data shown are from a single representative of three separate experiments. (B) The change of relative CaOx crystal growth was plotted in relation to the concentrations of IαI and its derivatives. Results are the mean of two experiments, carried out on triplicate assays; the bar is SD. Symbols are: (□) heavy chains of IαI; (•) IαI; (▵) HI-8; (▪) chondroitinase AC-treated UTI; (○) UTI. Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 SDS-PAGE followed by Western blot of the matrix proteins extracted from CaOx crystals generated in urine in the presence or absence of IαI or its derivatives. The proteins that are most prominent in the gel are the ones that reacted with polyclonal antibody against human prothrombin. The 31 kDa protein band can be seen; it is prothrombin fragment 1. Molecular weight standards (Bio-Rad) are shown on the far left. Western blot of the matrix protein extracted from CaOx crystals generated in the urine aliquots in the presence of IαI (lane 1), heavy chains (lane 2), UTI (lane 3), chondroitinase AC-treated UTI (lane 4), or HI-8 (lane 5). We analyzed the quantification of the 31 kDa protein band of each gel by scanning densitometry. Lane 1, 100%; lane 2, 89%; lane 3, 5%; lane 4, 20%; and lane 5, 41%. Kidney International 1998 53, 1727-1735DOI: (10.1046/j.1523-1755.1998.00940.x) Copyright © 1998 International Society of Nephrology Terms and Conditions