Hyaluronan Structures Synthesized by Rat Mesangial Cells in Response to Hyperglycemia Induce Monocyte Adhesion
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m312045200
ISSN1083-351X
Autores Tópico(s)Cell Adhesion Molecules Research
ResumoMesangial expansion, the principal glomerular lesion in diabetic nephropathy, is preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells and by a prominent glomerular infiltration of monocytes and macrophages. Because this infiltration seems to play a key role in the subsequent mesangial matrix expansion, we tested the response of cultures of rat mesangial cells (RMCs) for monocyte adhesion in response to hyperglycemia. Increasing the medium glucose concentration from 5.6 mm (normal) to 25.6 mm (hyperglycemic) significantly increased hyaluronan in the cell matrix, with a concurrent 3- to 4-fold increase in adhesion of U937 monocytic leukemic cells to cultures of near confluent RMCs. These responses were attributed directly to the high glucose concentration and not to increased extracellular osmolality. The monocytes primarily bind directly to hyaluronan-based structures in vitro. Abnormal deposits of hyaluronan were found in glomeruli of kidney sections from diabetic rats 1 week after streptozotocin treatment, often with closely associated monocytes/macrophages, suggesting that similar structures are relevant in vivo. The monocyte adhesion response to high glucose concentration required growth stimulation of RMCs by serum and activation of protein kinase C, and was inhibited by prior passage of the RMCs in the presence of heparin. These results suggest that the response may be cell growth state and protein kinase C-dependent. When incubated with the viral mimetic, poly I:C, in the presence of normal glucose, heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated adhesion of monocytes, indicating that the two stimuli, high glucose and viral mimetic, induce the production of the hyaluronan structures that promote monocyte adhesion by distinctly different intracellular signaling mechanisms. Mesangial expansion, the principal glomerular lesion in diabetic nephropathy, is preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells and by a prominent glomerular infiltration of monocytes and macrophages. Because this infiltration seems to play a key role in the subsequent mesangial matrix expansion, we tested the response of cultures of rat mesangial cells (RMCs) for monocyte adhesion in response to hyperglycemia. Increasing the medium glucose concentration from 5.6 mm (normal) to 25.6 mm (hyperglycemic) significantly increased hyaluronan in the cell matrix, with a concurrent 3- to 4-fold increase in adhesion of U937 monocytic leukemic cells to cultures of near confluent RMCs. These responses were attributed directly to the high glucose concentration and not to increased extracellular osmolality. The monocytes primarily bind directly to hyaluronan-based structures in vitro. Abnormal deposits of hyaluronan were found in glomeruli of kidney sections from diabetic rats 1 week after streptozotocin treatment, often with closely associated monocytes/macrophages, suggesting that similar structures are relevant in vivo. The monocyte adhesion response to high glucose concentration required growth stimulation of RMCs by serum and activation of protein kinase C, and was inhibited by prior passage of the RMCs in the presence of heparin. These results suggest that the response may be cell growth state and protein kinase C-dependent. When incubated with the viral mimetic, poly I:C, in the presence of normal glucose, heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated adhesion of monocytes, indicating that the two stimuli, high glucose and viral mimetic, induce the production of the hyaluronan structures that promote monocyte adhesion by distinctly different intracellular signaling mechanisms. Mesangial expansion, the principal glomerular lesion in diabetic nephropathy, reduces the area for filtration and leads eventually to sclerosis and renal failure (1Mauer S.M. Kidney Int. 1994; 45: 612-622Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 2Wolf G. Ziyadeh F.N. Kidney Int. 1999; 56: 393-405Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). However, the expansion of the mesangial extracellular matrix (ECM) 1The abbreviations used are: ECM, mesangial extracellular matrix; PKC, protein kinase C; RMCs, rat mesangial cells; FBS, fetal bovine serum; FACE, fluorophore-assisted carbohydrate electrophoresis; PBS, phosphate-buffered saline; poly I:C, a synthetic double-stranded RNA; HABP, hyaluronan-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; TRITC, tetramethylrhodamine isothiocyanate.1The abbreviations used are: ECM, mesangial extracellular matrix; PKC, protein kinase C; RMCs, rat mesangial cells; FBS, fetal bovine serum; FACE, fluorophore-assisted carbohydrate electrophoresis; PBS, phosphate-buffered saline; poly I:C, a synthetic double-stranded RNA; HABP, hyaluronan-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; TRITC, tetramethylrhodamine isothiocyanate. and the sclerosis that characterize diabetic nephropathy are preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells. This is followed by a prominent glomerular infiltration of monocytes and macrophages (3Young B. Johnson R. Alpers C. Eng E. Floege J. Couser W. J. Am. Soc. Nephrol. 1992; 3: 770Google Scholar, 4Furuta T. Saito T. Ootaka T. Soma J. Obara K. Abe K. Yoshinaga K. Am. J. Kidney Dis. 1993; 21: 480-485Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 5Young B.A. Johnson R.J. Alpers C.E. Eng E. Gordon K. Floege J. Couser W.G. Seidel K. Kidney Int. 1995; 47: 935-944Abstract Full Text PDF PubMed Scopus (316) Google Scholar) that seems to play a key role in the subsequent mesangial matrix expansion, hypercellularity, and onset of proteinuria (6Diamond J.R. Pesek-Diamond I. Am. J. Physiol. 1991; 260: F779-F786PubMed Google Scholar, 7Mené P. Caenazzo C. Pugliese F. Cinotti G.A. D'Angelo A. Garbisa S. Gambaro G. Nephrol. Dial. Transplant. 2001; 16: 913-922Crossref PubMed Scopus (8) Google Scholar). Nevertheless, the molecular mechanisms underlying glomerular infiltration by monocytes and macrophages are still unclear. The interaction of monocytes with mesangial cells in culture has been studied previously using U937 cells, a monocytic leukemic cell line that is a widely accepted model for monocyte adhesion (7Mené P. Caenazzo C. Pugliese F. Cinotti G.A. D'Angelo A. Garbisa S. Gambaro G. Nephrol. Dial. Transplant. 2001; 16: 913-922Crossref PubMed Scopus (8) Google Scholar, 8Mené P. Pugliese F. Cinotti G.A. Kidney Int. 1996; 50: 417-423Abstract Full Text PDF PubMed Scopus (16) Google Scholar), and mesangial cell cultures incubated in media with high glucose concentrations bind more U937 cells (7Mené P. Caenazzo C. Pugliese F. Cinotti G.A. D'Angelo A. Garbisa S. Gambaro G. Nephrol. Dial. Transplant. 2001; 16: 913-922Crossref PubMed Scopus (8) Google Scholar). Generally, the interactions between monocytes and resident cells involve: (i) monocyte cell surface proteins, including CD44, β-2 leukocyte integrins (LFA-1, CR3, and p150/95), the β-1 integrin VLA-4, and l-selectin, and (ii) their ligands, such as VCAM-1 and ICAM-1, on resident cells (9Mené P. Fais S. Cinotti G.A. Pugliese F. Luttmann W. Thierauch K.H. Nephrol. Dial. Transplant. 1995; 10: 481-489Crossref PubMed Scopus (27) Google Scholar, 10Rodriguez-Iturbe B. Pons H. Herrera-Acosta J. Johnson R.J. Kidney Int. 2001; 59: 1626-1640Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Previously, we showed that monocytes preferentially bind, by means of a mechanism that involves CD44, to hyaluronan cable-like structures synthesized by smooth muscle cells isolated from human intestinal mucosa, after they have been treated with a virus or with the viral mimetic, poly I:C (a synthetic double-stranded RNA) (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 12de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Am. J. Path. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). This indicates that structures based on hyaluronan can mediate the adhesion of monocytes to resident cells. Previous studies have shown increased hyaluronan content in glomeruli isolated from diabetic animals (13Dunlop M.E. Clark S. Mahadevan P. Muggli E. Larkins R.G. Kidney Int. 1996; 50: 40-44Abstract Full Text PDF PubMed Scopus (18) Google Scholar) and in mesangial cells cultured in medium with a high glucose concentration (14Mahadevan P. Larkins R.G. Fraser J.R. Fosang A.J. Dunlop M.E. Diabetologia. 1995; 38: 298-305Crossref PubMed Scopus (58) Google Scholar). Exposure to high glucose concentrations also increases hyaluronan synthesis in renal proximal tubular cells (15Jones S. Jones S. Phillips A.O. Kidney Int. 2001; 59: 1739-1749Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), in renal interstitial fibroblasts (16Takeda M. Babazono T. Nitta K. Iwamoto Y. Metabolism. 2001; 50: 789-794Abstract Full Text PDF PubMed Scopus (25) Google Scholar), and in vascular smooth muscle cells (17Erikstrup C. Pedersen L.M. Heickendorff L. Ledet T. Rasmussen L.M. Eur. J. Endocrinol. 2001; 145: 193-198Crossref PubMed Scopus (39) Google Scholar), all of which are involved in normal kidney physiology. Experiments described in this report indicate that: (i) cultures of rat mesangial cells (RMCs) exposed to high glucose concentrations can produce hyaluronan structures that promote monocyte adhesion; (ii) this process depends on the growth state and protein kinase C (PKC) activation of the RMCs; (iii) hyaluronan deposits, often with closely associated monocytes/macrophages, are apparent in 1-week diabetic rat glomeruli; and (iv) RMCs passaged in the presence of heparin lose their ability to produce the adhesive hyaluronan structures in response to high glucose concentrations but continue to do so when incubated with the viral mimetic, poly I:C, in medium with normal glucose concentration. These results support the hypothesis that hyaluronan structures in the mesangial ECM, produced in response to hyperglycemia, bind to specific receptors on the monocyte surface, thereby promoting monocyte and macrophage adhesion to mesangial matrix in the early stages of diabetic nephropathy. Establishment of RMC Cultures and Induction of Diabetes in Rats— RMC cultures were established from isolated glomeruli and characterized as described previously (18Simonson M.S. Dunn M.J. Methods Enzymol. 1990; 187: 544-553Crossref PubMed Scopus (37) Google Scholar, 19Templeton D.M. J. Biol. Chem. 1990; 265: 21764-21770Abstract Full Text PDF PubMed Google Scholar). RMCs were used between passages 5 and 15 when they still contract in response to angiotensin II and endothelin, and they exhibit growth suppression in the presence of heparin (1 μg/ml), which are additional characteristics of mesangial cells (20Hegele R.G. Behar M. Katz A. Silverman M. Clin. Invest. Med. 1989; 12: 181-186PubMed Google Scholar, 21Mené P. Simonson M.S. Dunn M.J. Physiol. Rev. 1989; 69: 1347-1424Crossref PubMed Scopus (459) Google Scholar, 22Wang A. Fan M.Y. Templeton D.M. J. Cell. Physiol. 1994; 159: 295-310Crossref PubMed Scopus (29) Google Scholar). RMCs were cultured in RPMI 1640 medium containing 10% FBS and passaged at confluence by trypsinization for 5 min with a solution of 0.025% trypsin, 0.5 mm EDTA. To render cells quiescent (22Wang A. Fan M.Y. Templeton D.M. J. Cell. Physiol. 1994; 159: 295-310Crossref PubMed Scopus (29) Google Scholar), cultures at ∼40% confluence (∼2 × 104 cells/cm2) were washed with RPMI 1640 medium and placed in fresh medium containing 0.4% FBS for 48–72 h (yielding 70–80% confluent cultures). To prepare the heparin-pretreated cells, the RMCs were cultured for three or five passages in the presence of 10 μg/ml heparin (23Templeton D.M. Zhao Y. Fan M.Y. Cardiovasc. Res. 2000; 45: 503-512Crossref PubMed Scopus (10) Google Scholar). U937 cells were cultured in suspension in RPMI 1640 medium containing 5% FBS and passaged at a 1:5 ratio (∼2 × 105 cells/ml) every 48 h (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Diabetes was induced in ∼175-g male Sprague-Dawley rats using tail vein injections of 55 mg/kg streptozotocin (5Young B.A. Johnson R.J. Alpers C.E. Eng E. Gordon K. Floege J. Couser W.G. Seidel K. Kidney Int. 1995; 47: 935-944Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 24Klein D.J. Brown D.M. Oegema T.R. Diabetes. 1986; 35: 1130-1142Crossref PubMed Scopus (43) Google Scholar). All animals were fed standard laboratory diet. Blood was collected by tail-bleeding at day 3 after injection, and the blood glucose concentration was determined by using fluorophore-assisted carbohydrate electrophoresis (FACE) analyses to confirm the onset of diabetes. After 1 week, the kidneys were collected from both control and diabetic rats and fixed in 4% paraformaldehyde in PBS at 4 °C overnight for subsequent processing and sectioning for histological analyses (Histology Core Facility, Department of Biomedical Engineering, Cleveland Clinic Foundation). Immunohistochemistry—Thin sections of paraffin-embedded kidneys and methanol-fixed RMC cultures on coverslips were stained for hyaluronan with a hyaluronan-binding protein (HABP) (Seikagaku America), for nuclei with 4′,6-diamidino-2-phenylindole (DAPI), and in the sections, for monocytes/macrophages with anti-rat ED1 antibody, as described previously (5Young B.A. Johnson R.J. Alpers C.E. Eng E. Gordon K. Floege J. Couser W.G. Seidel K. Kidney Int. 1995; 47: 935-944Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 12de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Am. J. Path. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 25Mukhopadhyay D. Hascall V.C. Day A.J. Salustri A. Fulop C. Arch. Biochem. Biophys. 2001; 394: 173-181Crossref PubMed Scopus (106) Google Scholar, 26Radounikli A. Stahl R.A.K. Bergman L. Schoeppe W. Thaiss F. Nephrol. Dial. Transplant. 1995; 10: 185-190PubMed Google Scholar). Samples were treated with HABP at a 1:100 dilution and with anti-rat ED1 antibody at a 1:75 dilution, washed and treated with fluorescein isothiocyanate-streptavidin at 1:500 dilution and/or with anti-mouse IgG TRITC antibody at 1:200 dilution. Stained samples were mounted in VectaShield containing DAPI (Vector Laboratories) for staining the nuclei of cells. Confocal images of the samples were obtained with a Leica TCS-NT laser-scanning, confocal microscope equipped with krypton and argon lasers. Assay for Monocyte Adhesion (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 27DiCorleto P.E. de la Motte C.A. J. Clin. Investig. 1985; 75: 1153-1161Crossref PubMed Scopus (91) Google Scholar)—RMCs in 6-well plates were treated up to 72 h with 10% FBS and concentrations of 5.6–25.6 mm d-glucose. After washing gently three times with cold medium, U937 monocytes (1 ml, 5 × 106 cells/ml) were added to the cultures and then incubated at 4 °C for 1 h. The cultures were then washed by placing 1 ml of cold medium gently onto the cultures, followed by rocking 3–5 times and aspirating from the side. The adhesion was done at 4 °C as previously described in detail (27DiCorleto P.E. de la Motte C.A. J. Clin. Investig. 1985; 75: 1153-1161Crossref PubMed Scopus (91) Google Scholar). The low temperature is necessary to prevent further metabolic responses of the monocytes. This method is routinely used for leukocyte adhesion assays. After washing, the cell cultures were imaged by microscopy with a Polaroid digital camera (7Mené P. Caenazzo C. Pugliese F. Cinotti G.A. D'Angelo A. Garbisa S. Gambaro G. Nephrol. Dial. Transplant. 2001; 16: 913-922Crossref PubMed Scopus (8) Google Scholar), and the numbers of monocytes per culture were counted using Metamorph software. Each culture was equally divided into four regions, and a culture area for imaging was randomly picked in each region. Bovine testicular hyaluronidase treatments (15 min, 37 °C at 200 units/ml) of RMCs before monocyte incubation were used to determine the extent of the hyaluronan-mediated adhesion. Proteinase K Release of Glycosaminoglycans from Cells and Tissues (28Calabro A. Benavides M. Tammi M. Hascall V.C. Midura R.J. Glycobiology. 2000; 10: 273-281Crossref PubMed Scopus (194) Google Scholar–30Calabro A. Midura R. Wang A. West L. Plaas A. Hascall V.C. Osteoarthritis Cartilage. 2001; 9: S16-S22Abstract Full Text PDF PubMed Scopus (97) Google Scholar)—Cell cultures were incubated with proteinase K at 250 μg/ml in 0.1 m ammonium acetate, pH 7.0, for 3 h at 60 °C. The reaction was terminated by heating the samples at >95 °C for 3–5 min. Hyaluronidase and Chondroitinase ABC Digestion—Samples were incubated with streptococcal hyaluronidase (50 milliunit/ml) and chondroitinase ABC at 2 units/ml in 0.1 m ammonium acetate, pH 7.0, overnight at 37 °C to generate disaccharides from hyaluronan and chondroitin/dermatan sulfate. The reaction was terminated by heating the samples at >95 °C for 3–5 min. FACE Analysis of Reducing Saccharides—Sample digests were dried by centrifugal evaporation in microtubes and then subjected to reductive amination with 2-aminoacridone as described previously (28Calabro A. Benavides M. Tammi M. Hascall V.C. Midura R.J. Glycobiology. 2000; 10: 273-281Crossref PubMed Scopus (194) Google Scholar, 29Calabro A. Hascall V.C. Midura R.J. Glycobiology. 2000; 10: 283-293Crossref PubMed Scopus (158) Google Scholar). At the end of the incubation, the samples were each mixed with glycerol to 20%; 5-μl aliquots were then subjected to electrophoresis on Mono-Composition gels with Mono Running buffer (Glyko). Running conditions were 500 V at 4 °C in a cold room for 1 h. Gels were imaged on an Ultra Lum transilluminator (365 nm). Images were captured and analyzed by using a Quantix cooled charge-coupled device camera from Roper Scientific/Photometrics and the Gel-Pro Analyzer program version 3.0 (Media Cybernetics), respectively. Glucose-induced Hyaluronan Synthesis in RMCs—When pre-confluent RMC cultures were incubated in 0.4% serum for 48 h, ∼75% of the starved cells were arrested in G0/G1 phase, as shown previously by using flow cytometry, and ∼72% subsequently progress to S-phase by 18 h after serum stimulation (31Wang A. Templeton D.M. Kidney Int. 1996; 49: 437-448Abstract Full Text PDF PubMed Scopus (43) Google Scholar). Therefore, after 48 h of serum-starvation, quiescent RMCs re-enter the cell cycle with good synchrony upon stimulation. Triplicate cultures of quiescent, near-confluent, and serum-starved RMC cultures were incubated for 72 h with 10% FBS in the presence of different concentrations of glucose, or in 5.6 mm glucose with 20 mm mannitol as an osmotic control. The hyaluronan contents of the cell layers were assessed by FACE analysis as described under "Experimental Procedures." Quantitation of the hyaluronan disaccharide in FACE gels such as that shown in Fig. 1 indicates that glucose treatment causes a statistically significant increase in hyaluronan content in the cell layers for the 20.6 and 25.6 mm glucose cultures and a trend for an increase between 5.6 and 15.6 mm glucose cultures. Further, the synthesized hyaluronan induced by high (25.6 mm) glucose was formed into large cable-like structures in RMC cell layers (Fig. 2, C and D), reminiscent of the hyaluronan structures observed previously that mediate monocyte adhesion to poly I:C-treated smooth muscle cell cultures (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 12de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Am. J. Path. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). These structures were not present in RMC cultures incubated in normal glucose with or without mannitol as an osmotic control (Fig. 2, A and B).Fig. 2Confocal microscopic analysis of hyaluronan structures in RMC cultures. The serum-starved mesangial cells on coverslips were incubated 72 h with medium containing 10% FBS in the presence of normal (5.6 mm, panel A) or high (25.6 mm, panels C and D) glucose. Mannitol (20 mm) with 5.6 mm glucose was used as an osmotic control (B). Hyaluronan was stained with HABP (green), and nuclei were stained with DAPI (blue). There are large hyaluronan structures in the cultures treated with high glucose (panels A–C, ×40; panel D, ×63).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of Glucose Concentration on Monocyte Adhesion to Rat Mesangial Cells—In the experiment in Fig. 3, near-confluent and serum-starved RMC cultures were incubated for 72 h in medium with 10% FBS and normal (5.6 mm) or high (25.6 mm) glucose concentration or in 5.6 mm glucose with 20 mm mannitol. Monocyte adhesion was then assayed using the U937 monocytic leukemia cell line, as described previously (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Monocyte adhesion to RMCs incubated with 25.6 mm glucose (Fig. 3B) was dramatically increased compared with RMCs treated with 5.6 mm glucose (Fig. 3A) or with 20 mm mannitol in 5.6 mm glucose as an osmotic control (Fig. 3C). The presence of serum is also necessary as RMCs cultured in high glucose with limiting serum (0.5%) showed monocyte adhesion similar to the 5.6 mm glucose cultures (data not shown). The role of hyaluronan in the adhesion process was underscored by treatment of RMCs incubated with high glucose with testicular hyaluronidase before assessing monocyte adhesion (Fig. 3D), in which case monocyte adhesion was greatly diminished. At a higher magnification (Fig. 4A, arrow), the adhesive monocytes in the RMCs incubated with high glucose were often arrayed in bead-like strands that appear similar to the patterns observed for monocyte adhesion to hyaluronan cable structures synthesized by colon smooth muscle cells in response to viral stimuli (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 12de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Am. J. Path. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). They also often appear in clusters (Fig. 4A, arrowhead). Confocal microscopic analysis of high glucose-treated cultures stained for hyaluronan (green) and nuclei (blue) (Fig. 4, B and C) show these configurations more clearly. In each case, the monocyte nuclei (Fig. 4, arrowheads) are closely arrayed to hyaluronan adhesive structures. In separate experiments, we have shown that glomeruli isolated from diabetic rats 1 week after treatment with streptozotocin have 2–3 times as much hyaluronan as glomeruli from control rats using the FACE procedures. 2M. Lauer, V. C. Hascall, A. Wang, unpublished data. This result would be coincident with the influx of monocytes/macrophages into the diabetic glomeruli (5Young B.A. Johnson R.J. Alpers C.E. Eng E. Gordon K. Floege J. Couser W.G. Seidel K. Kidney Int. 1995; 47: 935-944Abstract Full Text PDF PubMed Scopus (316) Google Scholar). Fig. 4 shows kidney tissue sections from both 1-week control and diabetic rats stained for hyaluronan (green) and monocytes/macrophages (red). They clearly demonstrate an abnormal accumulation of hyaluronan in the glomeruli of diabetic rats (compare Fig. 4D with E and F), with glomerular infiltrated monocytes/macrophages often embedded in this hyaluronan-enriched matrix (Fig. 4F, arrowheads). These results suggest that a causal link exists between glomerular hyaluronan accumulation and monocyte/macrophage infiltration, and that the hyaluronan structures may mediate monocyte adhesion and activation in diabetic nephropathy. Quantitation of Monocyte Adhesion—Triplicate serum-starved, near-confluent RMC cultures were incubated for 72 h with different concentrations of glucose (5.6–25.6 mm) in 10% FBS and then assessed for monocyte adhesion. The numbers of monocytes per unit area were then determined as described under "Experimental Procedures" (Fig. 5). Monocyte adhesion increased with exposure to increasing amounts of glucose, with statistical significance at concentrations of 15.6 mm and above. At 25.6 mm, there were more than 3 times as many adherent monocytes as observed with 5.6 mm glucose, and ∼75% of the difference was lost when RMC cultures exposed to 25.6 mm glucose were pretreated with hyaluronidase. Further, there is a correlation between increased hyaluronan content and increased adhesion of monocytes. The mannitol osmolarity control showed no difference from the normal (5.6 mm) glucose cultures. Further, pretreatment of RMC cultures exposed to normal (5.6 mm) glucose with hyaluronidase did not significantly alter the number of adherent monocytes (data not shown). Interestingly, the amount of hyaluronan in the cell layer of the RMC culture exposed to normal (5.6 mm) glucose was about half that of the 20.6 and 25.6 mm glucose cultures (Fig. 1), yet hyaluronan-mediated adhesion in the 5.6 mm glucose cultures was negligible. Thus, monocytes adhering to RMC cultures treated with normal glucose concentration did so by a mechanism independent of hyaluronan, possibly by means of a VCAM-1/VLA-4 mechanism (11de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The hyaluronan-based monocyte adhesion response described above occurs when quiescent preconfluent RMCs are stimulated to enter a mitotic cycle by replenishing serum, but in the presence of a high glucose concentration. In contrast, when confluent RMC cultures were treated identically, the number of adherent monocytes in cultures treated with 25.6 mm glucose did not increase significantly beyond the number adhering to preconfluent cultures in response to 5.6 mm glucose (Fig. 5C). This result suggests that the response is likely to be cell growth state dependent. Effects of Heparin on Glucose-induced Monocyte Adhesion to RMCs—Diabetic nephropathy in animal models is ameliorated when the animals are treated with heparin or heparan sulfate in vivo (32Gambaro G. Cavazzana A.O. Luzi P. Piccoli A. Borsatti A. Crepaldi G. Marchi E. Venturini A.P. Baggio B. Kidney Int. 1992; 42: 285-291Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 33Gambaro G. Venturini A.P. Noonan D.M. Fries W. Re G. Garbisa S. Milanesi C. Pesarini A. Borsatti A. Marchi E. Baggio B. Kidney Int. 1994; 46: 797-806Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Therefore, two different isolates of RMCs were passaged either 3 or 5 times in the presence or absence of 10 μg/ml heparin. Near-confluent RMC cultures were then prepared and exposed to 25.6 mm glucose in the presence of 10% serum and in the absence of heparin. In both isolates, RMCs passaged without heparin showed increased hyaluronan and adhesion of monocytes in response to high glucose treatment (Figs. 6 and 7, CON), as described above (Figs. 3 and 5). Conversely, RMCs passaged in the presence of heparin no longer responded to the high glucose treatment, neither in increased hyaluronan production in the cell layer nor in increased monocyte adhesion (Figs. 6 and 7). However, these heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated monocyte adhesion when incubated with the viral mimetic, poly I:C, in the presence of normal (5.6 mm) glucose (Figs. 6 and 7). RMCs passaged without heparin also produced hyaluronan and promoted monocyte adhesion when exposed to poly I:C in the same conditions (data not shown). These results indicate that: (i) RMCs undergo phenotypic changes that make them unresponsive to high glucose when passaged in the presence of heparin; and (ii) there are two completely different mechanisms (high glucose and viral stimulus) for inducing the production of the hyaluronan structures that promote monocyte adhesion.Fig. 7Analysis of monocyte adhesion to heparin-pretreated mesangial cells. A and B, RMCs were cultured for three (data shown) or five (data not shown) passages in the presence of 10 μg/ml heparin and then plated in the absence of heparin and in the presence of normal (5.6 mm, A) or high (25.6 mm, B) glucose for the monocyte adhesion assay. C, in some cultures in 5.6 mm glucose, ∼15 μg/ml of poly I:C was added in the last 24 h of incubation. The heparin pre-treatment inhibited high glucose-induced monocyte adhesion (B) but did not affect the poly I:C-induced monocyte adhesion in normal glucose concentration (C). The cell cultures were imaged by microscopy with a Polaroid digital camera, and the numbers of monocytes per culture area were counted using Metamorph software (D). RMC control cultures passaged without heparin (CON) still responded to the high glucose. Nearly identical results were observed in the second experiment (***, p < 0.001; n = 8).View Large Image Figure ViewerDownload Hi-res
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