Search

Urine contains several macromolecules that inhibit calcium oxalate (CaOx) crystallization. Among them is bikunin, the light chain of most of the inter-alpha-inhibitor (IalphaI) family of glycoproteins. This study aimed to verify whether bikunin and other members of the IalphaI family are synthesized in the kidneys or derived exclusively from the plasma. Proteins extracted from homogenized bovine kidney were applied successively to three chromatographic steps on DEAE-Sephacel, Sephacryl S-300, and Mono Q column. The inhibitory activity was assayed using a CaOx crystallization system. The presence of IalphaI-related proteins was determined by electrophoresis and Western blotting. The results showed that kidney extract contained a 125-kDa protein that cross-reacted with anti-IalphaI antibodies. This protein inhibited CaOx crystallization efficiently. According to its molecular weight and immunoreaction with anti-IalphaI antibody, the 125-kDa protein could be pre-alpha-inhibitor. The latter is known to encompass a heavy chain and bikunin, which may explain its inhibitory activity against CaOx crystallization. Consequently, we hypothesize that kidneys may produce some IalphaI-related proteins that are involved in the inhibition of stone formation.

Our earlier studies indicated that members of the inter-alpha-inhibitor (IalphaI) family of glycoproteins may play an important role in urolithiasis. Indeed bikunin, the light chain of IalphaI is a potent inhibitor of calcium oxalate crystallization. In order to understand this role, the distribution of IalphaI and its related proteins, as well as the expression of bikunin, were studied in normal and nephrolithic rats. In normal rats, IalphaI immunoreactivity was located mainly in proximal tubules. However, in nephrolithic rats, in addition to proximal tubules, the staining was intensively extended to tubules in the corticomedullary junction. Furthermore, by using polymerase chain reaction technique, we demonstrated that gene encoding for bikunin was activated in kidneys of nephrolithic rats. We have previously demonstrated increased staining for osteopontin in association with calcium oxalate crystal deposition in rat kidneys. Others have shown an increase in osteopontin production by renal epithelial cells on exposure to calcium oxalate crystals. Based on these observations we conclude that kidney cells possess an auto-defense system against calcium oxalate crystallization and stone formation in which members of the IalphaI family may be closely involved.

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-alpha-inhibitor (IalphaI) related proteins, alpha-1 microglobulin (alpha-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine.

The involvement of Tamm Horsfall protein (THP) in nephrolithiasis is currently under investigation in several laboratories. Although rat is a commonly used species as an in vivo model for such studies, there is only limited information available about the biochemical properties and excretion profile of THP in normal rats. In order to characterize rat THP, we purified and analyzed normal male rat THP, and compared it with normal human male urinary THP by gel electrophoresis. Both THPs migrated at approximately 90 KDa, and stained similarly for protein (Coomassie blue) as well as carbohydrates (periodic acid Schiff reagent). Compositional analysis revealed that rat THP was largely similar to human THP in amino acid and carbohydrate contents but showed differences in the individual sugar components from other mammals. There was considerable variation in the day-to-day urinary excretion of THP in normal rats, with values ranging from 552.96 micrograms to 2865.60 micrograms and a mean value of 1679.54 micrograms per 24 h. It was concluded from this study that rat THP did not contain any unusual biochemical components and was primarily similar to human THP in composition and mean urinary concentration.

Crystalluria was induced in male Sprague-Dawley rats by implanting potassium-oxalate-containing mini-osmotic pumps. Urine of all experimental animals had abundant crystals of calcium oxalate, hydroxypatite, struvite, and calcium phosphate. These crystals were similar in morphology to the crystals found in human crystalluria. Histological examination of kidneys and tissue samples from other organs revealed no abnormality. One group of animals was injected with sodium oxalate in addition to implantation of potassium-oxalate-containing mini-osmotic pumps. Bladder urine from these animals contained calcium oxalate microstones and their kidneys had deposits of calcium oxalate crystals.