Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene ( PKD1 ) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an ‫ف‬ 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys. ( J. Clin. Invest. 1996. 98:2674-2682.)

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The kidney plays an important role in the metabolism of proteins and peptides. Current evidence indicates that only the proximal tubule possesses the mechanism for degradation or transport of these substances and reabsorption of metabolic products. Proteins and large polypeptides filtered at the glomerulus are absorbed from proximal tubular fluid by luminal endocytosis into apical vacuoles. These fuse with primary lysosomes, where hydrolysis occurs followed by diffusion of metabolites out of the cells and into the blood. Recent evidence indicates that small linear peptides are handled by a different mechanism. It is likely that small peptides are degraded at the luminal surface of the brush border of proximal tubules, which contains many hydrolytic enzymes, by the process of membrane or contact digestion with reabsorption of the breakdown products. The probable biological significance of proximal tubular mechanisms for handling of proteins and peptides are conservation of amino acids, inactivation of toxic substances, and participation in the regulation of the circulating level of protein and peptide hormones.

Biosynthetic regulation of renal glomerular heparan sulfate-proteoglycans by various aldohexoses (mannose, glucose, and galactose) and laminin. Cellular ATP levels were dramatically reduced in all groups, and the maximal depletion was caused by mannose. Addition of ATP (0.1-1.0 mM) to the perfusion medium resulted in the normalization of the de novo synthesis and of the biochemical characteristics of heparan sulfate-proteoglycans. The relevance of decreased de novo synthesis of proteoglycans due to the depletion of ATP in hyperglycemic states is discussed in terms of increased glomerular permeability to plasma proteins, as seen in diabetes mellitus.During ultrafiltration, the glomerular basement membrane (GBM) restricts the passage of circulating plasma proteins into the urinary space (1). Thus, a compromise in the integrity of the GBM would be expected to lead to the leakage of proteins into the urine, as observed in a variety of renal diseases, including diabetic nephropathy (2, 3). In diabetes, there is a remarkable thickening of the GBM, and an increase in the mesangial matrices associated with proteinuria (4, 5). The GBM alterations may be due to the metabolic derangements in its various components-i.e., type IV collagen, laminin, entactin, and proteoglycans (PGs) (3, 6, 7). In this context, several studies have indicated a loss or a decrease in the synthesis of PGs in diabetes (3, 6-10). The loss of PGs has been regarded as one of the key events responsible for the proteinuric response seen in diabetes (4, 5). Although the deficiency of PGs/glycosaminoglycans (GAGs) is well documented, the mechanism(s) responsible for their decreased synthesis remains to be elucidated. This investigation relates to the study of one of the possible mechanisms-i.e., ATP depletion caused by high aldohexose levels-that may be responsible for the decreased de novo synthesis of heparan sulfate (HS)-PGs in diabetes. METHODSRadiolabeling of Glomerular PGs. An ex vivo organ perfusion system was utilized for radiolabeling of PGs with [35S]sulfate (100 ,uCi/ml; 1 ,uCi = 37 kBq) (11). The hexoses used in various experiments were mannose, glucose, and galactose. They were individually added (10-50 mM) to the perfusion medium. Mannitol (25 mM) was included in the medium as a control. After 5 hr of radiolabeling of the rat kidney, a small cortical piece was processed for tissue autoradiography and the remaining kidney was perfused with Hanks' balanced salts solution containing a mixture of protease inhibitors (11). The kidney was then bisected, the cortex was dissected out, and glomeruli were isolated. Both the isolated glomeruli and the perfusion medium were processed for isolation and characterization of PGs/GAGs.Isolation and Characterization of PGs/GAGs. The radiolabeled PGs from isolated glomeruli were extracted with 4 M guanidinium chloride containing a mixture of protease inhibitors (11). An aliquot of the extract was passed through a Sephadex G-25 column equilibrated with 4 M guanidinium chloride/0.05 M sodium acetate/0...

Receptor-like protooncogenes, with tyrosine kinase catalytic domains, are expressed in neoplastic and fetal tissues and potentially have a role in embryonic development. Which protooncogene may have the dominant role in embryonic renal development during the "postinductive" period, i.e., Day 10 onward, was addressed in this study by utilizing an in vitro organ culture system. The role of various receptor-like protooncogenes, with the emphasis on c-ros and c-ret, was investigated by antisense-oligodeoxynucleotide (ODN) gene-targeting strategies at a point in metanephric development when reciprocal-inductive interactions between the epithelium and mesenchyme have already been initiated and are rampant. Also, their relationship with other morphogens, like extracellular matrix (ECM) proteins and growth factors, was studied. Initial in situ hybridization and RT-PCR analyses revealed a similar spatiotemporal expression for both c-ros and c-ret in the embryonic kidneys. At Day 13, they were mainly expressed in the developing nephrons in the nephrogenic zone and ureteric bud branches, where the signals from the mesenchymal ligands are transduced to the epithelial cell surface receptors. Minimal expression was observed in the newborn kidneys. Inclusion of antisense ODNs, derived from the phosphotyrosine kinase domains, inhibited metanephric growth in the organ culture; the most dramatic effects were observed with the c-ret antisense ODN. The c-ret-induced dysmorphogenetic effects were characterized as a decrease in the population of nephrons, atrophy of the mesenchymal cells, and loss of acuteness of the tips of ureteric bud branches. Interestingly, the ureteric bud branches continue to grow in the atrophic mesenchyme. Both c-ros and c-ret antisense ODNs reduced the gene expression and biosynthesis of various ECM proteins. The proteoglycans, expressed at the epithelial:mesenchymal interface, were most adversely affected, especially by the c-ret antisense. The treatment of metanephric explants with c-ret did not affect the gene expression of c-ros and vice versa. The specificity of the effects of c-ret antisense was also reflected by a decrease of anti-Ret protein immunoreactivity. The studies were extended to establish a relationship between c-ret protooncogene and some of the growth factors which are known to influence renal development via their tyrosine kinase-like receptors localized in the ureteric bud branches, the site apparently where c-ret is also expressed. Among the various growth factors examined, transforming growth factor-alpha (TGF-alpha) and insulin like growth factor-I (IGF-I) had the most notable trophic effects on metanephric explants and caused maximal phosphorylation of Ret protein. In addition, concurrent exposure of TGF-alpha or IGF-I and c-ret antisense ODN explants caused partial recovery from the c-ret-induced dysmorphogenetic effects in the metanephroi. The data suggest that, although a number of protooncogenes share similar catalytic domains, c-ret plays a major role during the "postinductive" ...