Characterization of Tamm-Horsfall protein in a rat nephrolithiasis model.

PURPOSE: The role of Tamm-Horsfall protein in calcium oxalate stone formation is controversial. It is unclear whether Tamm-Horsfall protein has a role in crystallization. If it does, does it act as an inhibitor or promoter of crystallization? To elucidate the nature of its involvement we characterized Tamm-Horsfall protein in a rat model of calcium oxalate nephrolithiasis by in vivo and in vitro techniques. MATERIALS AND METHODS: Calcium oxalate nephrolithiasis was induced in male Sprague-Dawley rats. The amino acid and carbohydrate composition of Tamm-Horsfall protein from normal rats and those with nephrolithiasis was determined. The Tamm-Horsfall protein gene and protein expression in the kidneys were examined by in situ hybridization and immunohistochemistry. Furthermore, the interaction of Tamm-Horsfall protein and calcium oxalate crystals was assessed by an in vitro crystal aggregation assay. RESULTS: Tamm-Horsfall protein from rats with nephrolithiasis was biochemically similar to that from normal rats. Although Tamm-Horsfall protein was associated with crystal deposits in the renal papillae of rats with nephrolithiasis, Tamm-Horsfall protein messenger RNA expression in the kidneys remained unchanged. In each group Tamm-Horsfall protein inhibited calcium oxalate crystal aggregation by 47%, indicating no change in functional capabilities. CONCLUSIONS: The results of this study indicate that urinary excretion, and the biochemical nature and functional capabilities of Tamm-Horsfall protein remain unchanged during experimental calcium oxalate nephrolithiasis. Although staining for Tamm-Horsfall protein was evident in the papillae of rats with nephrolithiasis, the site of Tamm-Horsfall protein synthesis remained cells of the thick ascending limbs of the loop of Henle.

The cytoplasmic domain of human FcgammaRIa alters the functional properties of the FcgammaRI.gamma-chain receptor complex.

The gamma/zeta-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding alpha-chain alters the biological properties of the receptor complex. Using macrophage FcgammaRIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human FcgammaRIa. Both receptors functionally associate with the endogenous murine gamma-chain. However, we have established that the CY of FcgammaRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcgammaRIa CY leads to slower kinetics of receptor-specific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent FcgammaRIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates FcgammaRIa-specific secretion of interleukin-6 but does not affect production of interleukin-1beta. These results demonstrate a functional role for the CY of FcgammaRIa and provide a general model for understanding how multiple receptors that utilize the gamma-chain can generate diversity in function.

Biochemical and quantitative analysis of Tamm Horsfall protein in rats.

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.

Localization of tamm-horsfall protein and osteopontin in a rat nephrolithiasis model.

The possibility of more than one urinary protein being simultaneously associated with calcium oxalate (CaOx) crystallization in vivo was investigated by examining the localization of Tamm-Horsfall protein (THP) and osteopontin (Opn) in a rat model of nephrolithiasis. CaOx crystal deposits were induced in male Sprague-Dawley rats by feeding 0.75% ethylene glycol in drinking water. THP and Opn were localized on kidney sections by immunoperoxidase technique, using specific polyclonal antibodies. When only occasional crystal deposits were seen in the kidney, THP showed a similar to normal pattern of distribution, with positive staining in the thick ascending limbs of the loop of Henle. Opn was localized in some nephrons in the thin limb of loop of Henle and on the papillary surface in the calyceal fornix. In contrast, in samples with a significantly increased number of deposits in the kidneys, the staining for both THP and Opn was strikingly enhanced and altered, with positive staining around the crystals as well as abnormal localization in the papilla. Interestingly, the occurrence of Opn was, however, more consistent than that of THP. This is a first study showing that in this nephrolithiasis model, normal localization of THP and Opn is altered and they are closely and concurrently associated with crystal deposits in vivo.

Immunocytochemical localization of Tamm-Horsfall protein in the kidneys of normal and nephrolithic rats.

Studies using in vitro systems have indicated that Tamm-Horsfall protein (THP) can interact with calcium oxalate (CaOx) crystals during kidney stone formation. However, information regarding the nature of its participation in this process remains controversial and unclear. In order to better understand the putative interaction of THP and crystals in vivo, we compared the localization of THP in normal rats and in chronic and semi-acute rat models of nephrolithiasis. In these rats, CaOx crystal deposits were induced in the kidneys by administering ethylene glycol (EG) in drinking water. The formation of CaOx mono- and dihydrate aggregates in the urine was confirmed by scanning electron microscopy. Immunohistochemical localization, as well as protein A-gold labeling at the ultrastructural level, demonstrated that in addition to its normal distribution, THP specifically associated with the renal crystal deposits. The THP-containing, organic matrix-like material consisted of a fine, fibrillar meshwork surrounding individual crystals and their aggregates. In addition, THP also appeared in the papilla, where it is normally absent, concurrent with the appearance of crystal deposits in the kidneys. These observations indicate that in nephrolithic rats the normal localization of THP is altered. Such an alteration may indicate an important physiological event related to crystal aggregation and kidney stone formation.

Structure of rat kidneys following microwave accelerated fixation.

In contrast to fixation of tissue in externally heated fixative, microwave-irradiation can generate uniform internal heat, which is of utmost importance for successful fixation of biological tissue. To evaluate the effectiveness of microwave-accelerated chemical fixation, we compared the structure of rat kidney fixed by a conventional method and a microwave-accelerated method, by scanning and transmission electron microscopy. Following perfusion, rat kidney pieces 1-2 mm in size were irradiated in Karnovsky's fixative in a domestic Amana microwave oven, till the temperature of the fixative reached 45-50 degrees C. For conventional fixation, tissue pieces were fixed overnight at room temperature in the same fixative. Both types of samples were processed further for electron microscopy using identical protocols. The microwave fixed samples showed excellent preservation of structure comparable to the samples fixed by the conventional method. Glomeruli and the renal tubules showed normal morphology with no cellular swelling. The cytoplasm and nuclear matrix of the epithelial cells was uniformly dense. Other fixation sensitive organelles like mitochondria and Golgi apparatus showed superior preservation with continuous membranes. These results demonstrate that microwave accelerated chemical fixation results in excellent preservation of tissue structure, reduces processing time significantly and is therefore a practical alternative to conventional protocols.