heparitin-sulfate and Polycystic-Kidney-Diseases

Normal human renal epithelial cells (NK) and cells from cysts of autosomal-dominant polycystic kidneys (ADPKD) were radiolabeled with [35S]sulfate. A two- to three-fold decrease in the radioactivity incorporated into the proteoglycan (PG) fraction, as ascertained by tissue autoradiography and biochemical techniques, was observed in the ADPKD group. In subconfluent NK cells, PGs eluted as two peaks with different proportions of chondroitin sulfate (CS) and heparan sulfate (HS) in the cellular and media fractions. In the confluent stage, only a single major peak in the media and matrix fractions was seen and had variable proportions of CS and HS. In subconfluent ADPKD monolayers, cellular PGs eluted as two peaks, with the major peak of higher molecular weight compared with NK cells. In confluent stage, there was a single PG peak of a relatively higher molecular weight, with a variable increase in the proportions of CS vs. HS and lower charge-density characteristics. These findings indicate that size and species of PGs vary during subconfluent and confluent stages of culture and elucidate a defect in the biosynthesis of PGs in human ADPKD cells.

Topics: Autoradiography; Chondroitin Sulfates; Genes, Dominant; Heparitin Sulfate; Humans; Killer Cells, Natural; Polycystic Kidney Diseases; Precipitin Tests; Proteoglycans; Reference Values

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Epithelium; Heparan Sulfate Proteoglycans; Heparitin Sulfate; In Vitro Techniques; Kidney Tubules; Polycystic Kidney Diseases; Proteoglycans; Sulfur Radioisotopes; Swine

Basement membranes surround the renal tubules and have been shown to limit their distension in vitro. Therefore, it has been postulated that a defect in a basement membrane component(s) underlies the pathogenesis of polycystic kidney disease. Here we have studied a murine model of congenital polycystic kidney disease and found by immunohistology, that the components of the peri-cyst basement membrane appeared to diminish with time. We also measured mRNA levels for collagen IV and laminin, and found a different pattern than in the normal mouse kidney. In normal kidneys, mRNA levels for the B1 and B2 chains of laminin were maximal at birth, and at 1 week for the alpha 1(IV) chain of collagen IV. With all three chains, the levels then rapidly declined. In contrast, mRNA for the alpha 1(IV) chain in congenital polycystic kidneys was half normal 1 week after birth and then increased. Laminin B1 and B2 chain mRNA's were 80% of normal at 1 week but were maintained at that level. As a control, beta-actin mRNA was examined and found to remain constant in both normal and diseased kidneys. In situ hybridization of cRNA probes for the alpha 1(IV) chain confirmed that cells associated with cysts were the principal source of expression of these basement membrane mRNAs. Thus, there exists an abnormal regulation of basement membrane gene expression in congenital polycystic kidney disease. The first stage is characterized by reduced levels of expression. In the second stage, the levels are abnormally high, perhaps representing a compensatory synthesis of basement membrane as cysts enlarge.

Topics: Animals; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Disease Models, Animal; DNA; Fluorescent Antibody Technique; Gene Expression Regulation; Genes; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney; Laminin; Mice; Mice, Inbred C57BL; Nucleic Acid Hybridization; Organ Size; Polycystic Kidney Diseases; RNA, Messenger

Status of basement membrane antigens in renal polycystic disease was investigated. Antibodies directed against various components of basement membrane, including anti-heparan sulfate proteoglycan, Type IV collagen, laminin, and fibronectin, were employed. Their reactivities with basement membranes of normal and cystic segments of the renal tubules were ascertained by indirect immunofluorescence. The tissues were obtained either from kidneys of patients with adult (autosomal dominant) polycystic disease or from rats with renal cystic change induced by administration of 2-amino-4,5-diphenylthiazole HCl. The human and rat tissues that had undergone cystic change exhibited essentially similar results. A loss of reactivity to anti-heparan sulfate proteoglycan antibodies was observed. The reactivity toward anti-Type IV collagen and laminin either remained unchanged or was focally increased. The reactivity toward fibronectin, normally absent, increased dramatically in the peritubular regions and interstitium. The results indicate that there is an imbalance in various antigenic components associated with renal tubular cystic disease in rat and man, which may have a fundamental role in the pathogenesis of this disorder.

Topics: Animals; Antigens; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Fibronectins; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Polycystic Kidney Diseases; Rats