Alterations in Human Glomerular Epithelial Cells Interacting with Nonenzymatically Glycosylated Matrix

Abstract

The glomerular epithelial cells and the glomerular basement membrane are important constituents of the permselective barrier in the kidney. These are affected in diabetic nephropathy, one of the long-term complications in diabetic patients. Nonenzymatic glycosylation resulting in the accumulation of advanced glycosylation end products correlates with the development of long-term complications in diabetes. The interaction of cells with extracellular matrix proteins plays a critical role in a variety of biological processes. Recent studies show that cell-matrix interactions mediated by integrins can transduce biochemical signals to the cell interior and regulate cell behavior. In this paper we demonstrate that interactions of human glomerular epithelial cells with a nonenzymatically glycated matrix are altered with defective cell spreading, reduced phosphorylation of focal adhesion kinase and reduced activity of mitogen-activated protein kinase. These data suggest that matrix glycation interferes with normal cell-matrix interactions and intracellular signaling that can potentially result in differential gene expression contributing to the changes seen in diabetic nephropathy. The glomerular epithelial cells and the glomerular basement membrane are important constituents of the permselective barrier in the kidney. These are affected in diabetic nephropathy, one of the long-term complications in diabetic patients. Nonenzymatic glycosylation resulting in the accumulation of advanced glycosylation end products correlates with the development of long-term complications in diabetes. The interaction of cells with extracellular matrix proteins plays a critical role in a variety of biological processes. Recent studies show that cell-matrix interactions mediated by integrins can transduce biochemical signals to the cell interior and regulate cell behavior. In this paper we demonstrate that interactions of human glomerular epithelial cells with a nonenzymatically glycated matrix are altered with defective cell spreading, reduced phosphorylation of focal adhesion kinase and reduced activity of mitogen-activated protein kinase. These data suggest that matrix glycation interferes with normal cell-matrix interactions and intracellular signaling that can potentially result in differential gene expression contributing to the changes seen in diabetic nephropathy. The glomerular epithelial cells together with the glomerular basement membrane participate in the formation of the permselective barrier in the kidney. Thickening of the glomerular basement membrane and expansion of the mesangium are among the characteristic changes seen in diabetic nephropathy, and they correlate with declining renal function in diabetic patients (1Mauer S.M. Steffes M.W. Ellis E.N. Sutherland D.E.R. Brown D.M. Goetz F.C. J. Clin. Invest. 1984; 74: 1143-1155Crossref PubMed Scopus (1058) Google Scholar).In diabetes, hyperglycemia affects many metabolic pathways and each of these changes has important consequences in the development of various pathologic manifestations. There is much documented evidence, including that from The Diabetes Control and Complications Trial (2The Diabetes Control and Complications Trial Research Group N. Engl. J Med. 1993; 329: 977-979Crossref PubMed Scopus (22513) Google Scholar), that hyperglycemia and subsequent biochemical events correlate with the development of diabetic complications. One of the mechanisms by which elevated sugar levels compromise normal structure and function is by the phenomenon of nonenzymatic glycosylation with the formation of primary Amadori, intermediate and advanced glycosylation end products (3Brownlee M. Vlassara H. Cerami A. Ann. Intern. Med. 1984; 101: 527-550Crossref PubMed Scopus (906) Google Scholar) that alter the structure and function of basement membrane macromolecules in vitro and in vivo (4Tsilibary E.C. Charonis A.S. Reger L.A. Woulhueter R.M. Furcht L.T. J. Biol. Chem. 1988; 263: 4302-4308Abstract Full Text PDF PubMed Google Scholar, 5Anderson S.S. Charonis A.S. Kim Y. Tsilibary E.C. J. Am. Soc. Nephrol. 1992; 3: 625Google Scholar, 6Charonis A.S. Tsilibary E.C. Diabetes. 1992; 41: 49-51Crossref PubMed Google Scholar). The accumulation of these products correlates with the development of long-term complications in diabetes (7Monnier V.M. Vishwanath V. Frank K.E. Elmets C.A. Douchat P. Kohn R.R. N. Engl. J. Med. 1986; 314: 403-408Crossref PubMed Scopus (533) Google Scholar).It is well accepted that extracellular matrices, especially basement membranes, are involved in a dynamic and reciprocal relationship with cells and to a large extent determine cellular phenotype, function, and gene expression (8Bisell M.J. Barcellos-Hoff M. J. Cell Sci. (Suppl.). 1987; 8: 327-335Crossref PubMed Google Scholar). These interactions of cells with matrix molecules are mediated largely by cell receptors of the integrin family (9Rouslahti E. J. Clin. Invest. 1991; 87: 1-5Crossref PubMed Scopus (1478) Google Scholar, 10Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar, 11Humphries M.J. Mould A.P. Tuckwell D.S. Bioessays. 1993; 15: 391-397Crossref PubMed Scopus (54) Google Scholar). The integrins form clusters at sites of close cell-substrate contact termed focal contacts, and these serve to link the integrins to the cytoskeleton. However, focal contacts play more than a structural role; they serve as centers for the activation of several signal transduction pathways (12Kornberg L.J. Earp H.S. Turner C.E. Prockop C. Juliano R.L. Proc. Natl Acad Sci. U. S. A. 1991; 88: 8392-8396Crossref PubMed Scopus (630) Google Scholar, 13Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Crossref PubMed Scopus (1527) Google Scholar, 14Clark E.A. Brugge J.S. Science. 1995; 268: 233-238Crossref PubMed Scopus (2809) Google Scholar). Tyrosine phosphorylation is a common and ubiquitous response to integrin activation in many cell types including leukocytes, fibroblasts, and carcinoma cells. Tyrosine phosphorylation of important components of the focal adhesion complex such as focal adhesion kinase (FAK) 1The abbreviations used are: FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; tIV, type IV collagen; tIV-C, control tIV; BSA, bovine serum albumin; HGEC, human glomerular epithelial cell; ANOVA, analysis of variance. 1The abbreviations used are: FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; tIV, type IV collagen; tIV-C, control tIV; BSA, bovine serum albumin; HGEC, human glomerular epithelial cell; ANOVA, analysis of variance. and paxillin, occurs following integrin activation (15Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 16Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-235Crossref PubMed Scopus (256) Google Scholar). Integrin engagement has also been linked to activation of MAPK (17Chen Q. Kinch M.S. Ling T.H. Burridge K. Juliano R.L. J. Biol. Chem. 1994; 269: 26602-26605Abstract Full Text PDF PubMed Google Scholar, 18Schlaepfer D.D. Hanks S.K. Hunter T. Geer P.V. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1438) Google Scholar). AP-1 is a sequence-specific transcriptional activator, and its activity is regulated by mitogen-activated protein kinases (19Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). Thus, one can see how integrin-mediated cell interactions with the extracellular matrix influence cell behavior and gene expression.In this work, we examine the possibility that interactions of cells with a diabetically modified matrix interfere with normal cell-matrix interactions and consequently result in changes in cell behavior. We have investigated the alterations seen when human glomerular epithelial cells interact with glycated matrix. This provides an insight into the role played by the human glomerular epithelial cells in the pathogenesis of diabetic nephropathy.DISCUSSIONThe mechanisms by which hyperglycemia results in altered cell behavior in vitro or in situ are multiple and complex. One of the mechanisms is nonenzymatic glycosylation, which is known to alter the structural and functional integrity of extracellular matrix. In the present report we provide evidence that these matrix modifications additionally affect cell-matrix interactions and behavior of human glomerular epithelial cells.As cells adhere and spread they form focal adhesions. Focal adhesions are sites of integrin clustering and activation of several signal transduction pathways (16Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-235Crossref PubMed Scopus (256) Google Scholar). It has been proposed that they may integrate mechanical signals associated with changes in cell shape with chemical signals elicited directly by integrin binding and thereby modulate downstream signaling (35Ingber D.E. Annu. Rev. Physiol. 1997; 59: 575-597Crossref PubMed Scopus (1311) Google Scholar). With increased cell spreading the total area of cell-extracellular matrix contact increases, hence focal adhesion formation, integrin binding, and therefore subsequent signaling events may vary in parallel. In addition it has been recently demonstrated that cell shape by itself is an important determinant of cell functions like survival and growth (36Chen C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4145) Google Scholar). Therefore alterations in cell spreading could both directly and indirectly affect normal cell responses.Defects in cell spreading have been noted on interaction of mesangial cells and endothelial cells with a glycated matrix (29Anderson S.S. Kim Y. Tsilibary E.C. Kidney Int. 1994; 46: 1359-1367Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 30Hasegawa G. Hunter A. Charonis A.S. J. Biol. Chem. 1995; 270: 3278-3283Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and changes in intracellular signaling have been observed under diabetic conditions in mesangial cells and endothelial cells (30Hasegawa G. Hunter A. Charonis A.S. J. Biol. Chem. 1995; 270: 3278-3283Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 37Cosio F.G. Wilmer W.A. Kidney Int. 1995; 48 ($$): 12-17Google Scholar). However, to the best of our knowledge there is no report of such studies in human glomerular epithelial cells in diabetic conditions, and changes in MAPK activation have not been investigated in these earlier reports.In diabetic nephropathy some of the changes occurring in the glomerulus are a thickening of the glomerular basement membrane and expansion of the mesangial matrix. The thickening of the glomerular basement membrane and the expansion of the mesangial matrix are due to an increased deposition of extracellular matrix macromolecules such as collagen, fibronectin, laminin, and proteoglycans (38Osterby R. Gunderson H.J.G. Diabetologia. 1980; 18: 493-500Crossref PubMed Scopus (129) Google Scholar, 39Abrass C.K. Peterson C.V. Raugi G.J. Diabetes. 1988; 37: 1695-1702Crossref PubMed Google Scholar, 40Ledbetter S. Copeland E.J. Noonan D. Vogeli G. Hassell J.R. Diabetes. 1990; 39: 196-203Crossref PubMed Google Scholar, 41Ayo S.H. Radnik R.A. Glass W.F. Garoni J.A. Rampt E.R. Appling D.R. Kreisberg J.I. Am. J. Physiol. 1991; 260: F185-F191PubMed Google Scholar, 42Yasuda T. Kohara Y. Usui T Kondo S. Harris R.C. Owada S. Ishida M. J. Am. Soc. Nephrol. 1996; 7 (Abstr. 3169): 1880Google Scholar). It has been seen that this could be due to an imbalance between matrix deposition and degradation. In streptozotocin-induced diabetes in rats, there is an up-regulation of expression of collagens, a down-regulation of the expression of collagenases, and an up-regulation of the expression of tissue inhibitors of collagenases (43Border W. Okuda S. Nakamura T. Semin. Nephrol. 1989; 9: 307-317PubMed Google Scholar).AP-1 is a sequence-specific transcriptional activator composed of members of the Jun and Fos families, and its activity is regulated by Mitogen activated protein kinases (19Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). In addition, it has been demonstrated that the AP-1 binding site regulates the transcription of the collagenase gene in response to integrin-mediated signals (44Tremble P. Damsky C.H. Werb Z. J. Cell Biol. 1995; 29: 1707-1720Crossref Scopus (115) Google Scholar). Therefore reduced MAPK activity could potentially cause alterations in the expression and secretion of matrix-degrading enzymes that we are currently investigating.Altered cell-matrix interactions followed by alterations in intracellular signaling could translate downstream into differential expression of matrix components, matrix degrading enzymes, and/or their inhibitors, growth factors, and that could to the changes occurring in a long-term diabetic The glomerular epithelial cells together with the glomerular basement membrane participate in the formation of the permselective barrier in the kidney. Thickening of the glomerular basement membrane and expansion of the mesangium are among the characteristic changes seen in diabetic nephropathy, and they correlate with declining renal function in diabetic patients (1Mauer S.M. Steffes M.W. Ellis E.N. Sutherland D.E.R. Brown D.M. Goetz F.C. J. Clin. Invest. 1984; 74: 1143-1155Crossref PubMed Scopus (1058) Google Scholar). In diabetes, hyperglycemia affects many metabolic pathways and each of these changes has important consequences in the development of various pathologic manifestations. There is much documented evidence, including that from The Diabetes Control and Complications Trial (2The Diabetes Control and Complications Trial Research Group N. Engl. J Med. 1993; 329: 977-979Crossref PubMed Scopus (22513) Google Scholar), that hyperglycemia and subsequent biochemical events correlate with the development of diabetic complications. One of the mechanisms by which elevated sugar levels compromise normal structure and function is by the phenomenon of nonenzymatic glycosylation with the formation of primary Amadori, intermediate and advanced glycosylation end products (3Brownlee M. Vlassara H. Cerami A. Ann. Intern. Med. 1984; 101: 527-550Crossref PubMed Scopus (906) Google Scholar) that alter the structure and function of basement membrane macromolecules in vitro and in vivo (4Tsilibary E.C. Charonis A.S. Reger L.A. Woulhueter R.M. Furcht L.T. J. Biol. Chem. 1988; 263: 4302-4308Abstract Full Text PDF PubMed Google Scholar, 5Anderson S.S. Charonis A.S. Kim Y. Tsilibary E.C. J. Am. Soc. Nephrol. 1992; 3: 625Google Scholar, 6Charonis A.S. Tsilibary E.C. Diabetes. 1992; 41: 49-51Crossref PubMed Google Scholar). The accumulation of these products correlates with the development of long-term complications in diabetes (7Monnier V.M. Vishwanath V. Frank K.E. Elmets C.A. Douchat P. Kohn R.R. N. Engl. J. Med. 1986; 314: 403-408Crossref PubMed Scopus (533) Google Scholar). It is well accepted that extracellular matrices, especially basement membranes, are involved in a dynamic and reciprocal relationship with cells and to a large extent determine cellular phenotype, function, and gene expression (8Bisell M.J. Barcellos-Hoff M. J. Cell Sci. (Suppl.). 1987; 8: 327-335Crossref PubMed Google Scholar). These interactions of cells with matrix molecules are mediated largely by cell receptors of the integrin family (9Rouslahti E. J. Clin. Invest. 1991; 87: 1-5Crossref PubMed Scopus (1478) Google Scholar, 10Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar, 11Humphries M.J. Mould A.P. Tuckwell D.S. Bioessays. 1993; 15: 391-397Crossref PubMed Scopus (54) Google Scholar). The integrins form clusters at sites of close cell-substrate contact termed focal contacts, and these serve to link the integrins to the cytoskeleton. However, focal contacts play more than a structural role; they serve as centers for the activation of several signal transduction pathways (12Kornberg L.J. Earp H.S. Turner C.E. Prockop C. Juliano R.L. Proc. Natl Acad Sci. U. S. A. 1991; 88: 8392-8396Crossref PubMed Scopus (630) Google Scholar, 13Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Crossref PubMed Scopus (1527) Google Scholar, 14Clark E.A. Brugge J.S. Science. 1995; 268: 233-238Crossref PubMed Scopus (2809) Google Scholar). Tyrosine phosphorylation is a common and ubiquitous response to integrin activation in many cell types including leukocytes, fibroblasts, and carcinoma cells. Tyrosine phosphorylation of important components of the focal adhesion complex such as focal adhesion kinase (FAK) 1The abbreviations used are: FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; tIV, type IV collagen; tIV-C, control tIV; BSA, bovine serum albumin; HGEC, human glomerular epithelial cell; ANOVA, analysis of variance. 1The abbreviations used are: FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; tIV, type IV collagen; tIV-C, control tIV; BSA, bovine serum albumin; HGEC, human glomerular epithelial cell; ANOVA, analysis of variance. and paxillin, occurs following integrin activation (15Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 16Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-235Crossref PubMed Scopus (256) Google Scholar). Integrin engagement has also been linked to activation of MAPK (17Chen Q. Kinch M.S. Ling T.H. Burridge K. Juliano R.L. J. Biol. Chem. 1994; 269: 26602-26605Abstract Full Text PDF PubMed Google Scholar, 18Schlaepfer D.D. Hanks S.K. Hunter T. Geer P.V. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1438) Google Scholar). AP-1 is a sequence-specific transcriptional activator, and its activity is regulated by mitogen-activated protein kinases (19Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). Thus, one can see how integrin-mediated cell interactions with the extracellular matrix influence cell behavior and gene In this work, we examine the possibility that interactions of cells with a diabetically modified matrix interfere with normal cell-matrix interactions and consequently result in changes in cell behavior. We have investigated the alterations seen when human glomerular epithelial cells interact with glycated matrix. This provides an insight into the role played by the human glomerular epithelial cells in the pathogenesis of diabetic nephropathy. mechanisms by which hyperglycemia results in altered cell behavior in vitro or in situ are multiple and complex. One of the mechanisms is nonenzymatic glycosylation, which is known to alter the structural and functional integrity of extracellular matrix. In the present report we provide evidence that these matrix modifications additionally affect cell-matrix interactions and behavior of human glomerular epithelial cells.As cells adhere and spread they form focal adhesions. Focal adhesions are sites of integrin clustering and activation of several signal transduction pathways (16Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-235Crossref PubMed Scopus (256) Google Scholar). It has been proposed that they may integrate mechanical signals associated with changes in cell shape with chemical signals elicited directly by integrin binding and thereby modulate downstream signaling (35Ingber D.E. Annu. Rev. Physiol. 1997; 59: 575-597Crossref PubMed Scopus (1311) Google Scholar). With increased cell spreading the total area of cell-extracellular matrix contact increases, hence focal adhesion formation, integrin binding, and therefore subsequent signaling events may vary in parallel. In addition it has been recently demonstrated that cell shape by itself is an important determinant of cell functions like survival and growth (36Chen C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4145) Google Scholar). Therefore alterations in cell spreading could both directly and indirectly affect normal cell responses.Defects in cell spreading have been noted on interaction of mesangial cells and endothelial cells with a glycated matrix (29Anderson S.S. Kim Y. Tsilibary E.C. Kidney Int. 1994; 46: 1359-1367Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 30Hasegawa G. Hunter A. Charonis A.S. J. Biol. Chem. 1995; 270: 3278-3283Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and changes in intracellular signaling have been observed under diabetic conditions in mesangial cells and endothelial cells (30Hasegawa G. Hunter A. Charonis A.S. J. Biol. Chem. 1995; 270: 3278-3283Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 37Cosio F.G. Wilmer W.A. Kidney Int. 1995; 48 ($$): 12-17Google Scholar). However, to the best of our knowledge there is no report of such studies in human glomerular epithelial cells in diabetic conditions, and changes in MAPK activation have not been investigated in these earlier reports.In diabetic nephropathy some of the changes occurring in the glomerulus are a thickening of the glomerular basement membrane and expansion of the mesangial matrix. The thickening of the glomerular basement membrane and the expansion of the mesangial matrix are due to an increased deposition of extracellular matrix macromolecules such as collagen, fibronectin, laminin, and proteoglycans (38Osterby R. Gunderson H.J.G. Diabetologia. 1980; 18: 493-500Crossref PubMed Scopus (129) Google Scholar, 39Abrass C.K. Peterson C.V. Raugi G.J. Diabetes. 1988; 37: 1695-1702Crossref PubMed Google Scholar, 40Ledbetter S. Copeland E.J. Noonan D. Vogeli G. Hassell J.R. Diabetes. 1990; 39: 196-203Crossref PubMed Google Scholar, 41Ayo S.H. Radnik R.A. Glass W.F. Garoni J.A. Rampt E.R. Appling D.R. Kreisberg J.I. Am. J. Physiol. 1991; 260: F185-F191PubMed Google Scholar, 42Yasuda T. Kohara Y. Usui T Kondo S. Harris R.C. Owada S. Ishida M. J. Am. Soc. Nephrol. 1996; 7 (Abstr. 3169): 1880Google Scholar). It has been seen that this could be due to an imbalance between matrix deposition and degradation. In streptozotocin-induced diabetes in rats, there is an up-regulation of expression of collagens, a down-regulation of the expression of collagenases, and an up-regulation of the expression of tissue inhibitors of collagenases (43Border W. Okuda S. Nakamura T. Semin. Nephrol. 1989; 9: 307-317PubMed Google Scholar).AP-1 is a sequence-specific transcriptional activator composed of members of the Jun and Fos families, and its activity is regulated by Mitogen activated protein kinases (19Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). In addition, it has been demonstrated that the AP-1 binding site regulates the transcription of the collagenase gene in response to integrin-mediated signals (44Tremble P. Damsky C.H. Werb Z. J. Cell Biol. 1995; 29: 1707-1720Crossref Scopus (115) Google Scholar). Therefore reduced MAPK activity could potentially cause alterations in the expression and secretion of matrix-degrading enzymes that we are currently investigating.Altered cell-matrix interactions followed by alterations in intracellular signaling could translate downstream into differential expression of matrix components, matrix degrading enzymes, and/or their inhibitors, growth factors, and that could to the changes occurring in a long-term diabetic The mechanisms by which hyperglycemia results in altered cell behavior in vitro or in situ are multiple and complex. One of the mechanisms is nonenzymatic glycosylation, which is known to alter the structural and functional integrity of extracellular matrix. In the present report we provide evidence that these matrix modifications additionally affect cell-matrix interactions and behavior of human glomerular epithelial cells. cells adhere and spread they form focal adhesions. Focal adhesions are sites of integrin clustering and activation of several signal transduction pathways (16Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-235Crossref PubMed Scopus (256) Google Scholar). It has been proposed that they may integrate mechanical signals associated with changes in cell shape with chemical signals elicited directly by integrin binding and thereby modulate downstream signaling (35Ingber D.E. Annu. Rev. Physiol. 1997; 59: 575-597Crossref PubMed Scopus (1311) Google Scholar). With increased cell spreading the total area of cell-extracellular matrix contact increases, hence focal adhesion formation, integrin binding, and therefore subsequent signaling events may vary in parallel. In addition it has been recently demonstrated that cell shape by itself is an important determinant of cell functions like survival and growth (36Chen C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4145) Google Scholar). Therefore alterations in cell spreading could both directly and indirectly affect normal cell in cell spreading have been noted on interaction of mesangial cells and endothelial cells with a glycated matrix (29Anderson S.S. Kim Y. Tsilibary E.C. Kidney Int. 1994; 46: 1359-1367Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 30Hasegawa G. Hunter A. Charonis A.S. J. Biol. Chem. 1995; 270: 3278-3283Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and changes in intracellular signaling have been observed under diabetic conditions in mesangial cells and endothelial cells (30Hasegawa G. Hunter A. Charonis A.S. J. Biol. Chem. 1995; 270: 3278-3283Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 37Cosio F.G. Wilmer W.A. Kidney Int. 1995; 48 ($$): 12-17Google Scholar). However, to the best of our knowledge there is no report of such studies in human glomerular epithelial cells in diabetic conditions, and changes in MAPK activation have not been investigated in these earlier In diabetic nephropathy some of the changes occurring in the glomerulus are a thickening of the glomerular basement membrane and expansion of the mesangial matrix. The thickening of the glomerular basement membrane and the expansion of the mesangial matrix are due to an increased deposition of extracellular matrix macromolecules such as collagen, fibronectin, laminin, and proteoglycans (38Osterby R. Gunderson H.J.G. Diabetologia. 1980; 18: 493-500Crossref PubMed Scopus (129) Google Scholar, 39Abrass C.K. Peterson C.V. Raugi G.J. Diabetes. 1988; 37: 1695-1702Crossref PubMed Google Scholar, 40Ledbetter S. Copeland E.J. Noonan D. Vogeli G. Hassell J.R. Diabetes. 1990; 39: 196-203Crossref PubMed Google Scholar, 41Ayo S.H. Radnik R.A. Glass W.F. Garoni J.A. Rampt E.R. Appling D.R. Kreisberg J.I. Am. J. Physiol. 1991; 260: F185-F191PubMed Google Scholar, 42Yasuda T. Kohara Y. Usui T Kondo S. Harris R.C. Owada S. Ishida M. J. Am. Soc. Nephrol. 1996; 7 (Abstr. 3169): 1880Google Scholar). It has been seen that this could be due to an imbalance between matrix deposition and degradation. In streptozotocin-induced diabetes in rats, there is an up-regulation of expression of collagens, a down-regulation of the expression of collagenases, and an up-regulation of the expression of tissue inhibitors of collagenases (43Border W. Okuda S. Nakamura T. Semin. Nephrol. 1989; 9: 307-317PubMed Google Scholar). AP-1 is a sequence-specific transcriptional activator composed of members of the Jun and Fos families, and its activity is regulated by Mitogen activated protein kinases (19Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). In addition, it has been demonstrated that the AP-1 binding site regulates the transcription of the collagenase gene in response to integrin-mediated signals (44Tremble P. Damsky C.H. Werb Z. J. Cell Biol. 1995; 29: 1707-1720Crossref Scopus (115) Google Scholar). Therefore reduced MAPK activity could potentially cause alterations in the expression and secretion of matrix-degrading enzymes that we are currently cell-matrix interactions followed by alterations in intracellular signaling could translate downstream into differential expression of matrix components, matrix degrading enzymes, and/or their inhibitors, growth factors, and that could to the changes occurring in a long-term diabetic We for the of Charonis for and for