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Glucose-induced overproduction of type IV collagen in cultured glomerular mesangial cells
Glucose-Induced Overproduction in Cultured Glomerular Mesangial
Masakazu Haneda Ryuichi Kikkawa Naoki Horide Masaki Togawa Daisuke Koya Nobuyuki Kajiwara Shiro Maeda Yukio Shigeta Third Department of Medicine, Shiga University of Medical Science, Shiga, Japan
of Type Cells
IV Collagen
ABSTRACT Type IV collagen production by cultured rat glomerular mesangial cells was evaluated quantitatively by measuring type IV collagen secreted into culture media and associated with the cells using enzymelinked immunosorbent assay (ELISA). The majority of type IV collagen was secreted into culture media; type IV collagen increased with cell growth in the early log phase and decreased in the late log phase and after cofluency. By exposing the cells to high concentrations of glucose (27.8 mhl), both secreted and cell-associated type IV collagens increased significantly compared with the cells cultured under normal glucose concentrations (5.6 mM) or under equivalent concentrations of mannitol, resulting in a significant increase in total type IV collagen accumulation. (The Journal of Diabetic Complications 5;23:199-200, 1991.)
INTRODUCTION Expansion of glomerular mesangium has been considered to be one of the major histological characteristics in diabetic nephropathy.’ Glomerular mesangial cells have been reported capable of synthesizing various collagens including type IV collagen2 one of the major components of the mesangial matrix. Therefore, if the production of type IV collagen by mesangial cells is enhanced in diabetes, it could accumulate in the mesangial area, leading to mesangial expansion. In order to prove this hypothesis, we examined the ability of mesangial cells to produce type IV collagen under high-glucose conditions by measuring it quantitatively with enzyme-linked immunosorbent assay (ELISA).
MATERIALSAND METHODS Mesangial Cell Culture Mesangial cells were obtained from the culture of glomeruli isolated from Sprague-Dawley rats as previously described.314 Mesangial cells, plated on a 60-mm culture dish in a density of 77,000 cells per dish, were cultured with RPM1 1640 media containing 20% fetal calf serum (FCS), 50 pg/ml ascorbic acid, and 50 pg/ml paminopropionitrile under the following conditions: (1) 5.6 mM glucose, (2) 27.8 mM glucose, and (3) 5.6 mM glucose plus 22.2 mM mannitol. Media were changed on days 4, 6, 8, and 12, and stored at -20°C until the assay. Cells were washed twice with phosphate-buffered saline (PBS), incubated with PBS containing 0.05% Triton X-100 at 4°C overnight, sonicated, and stored at -20°C. Reprint requests to be sent to: Masakazu Haneda, M.D., Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21, Japan.
0 1991 Elsevier Science Publishing Co., Inc 0891-6632/91/$3,50
Enzyme-Linked lmmunosorbent Assay of Type IV Collagen Type IV COIlagen was isolated from the lungs and kidneys of 20 Sprague-Dawley rats using limited pepsin digestion and differential salt precipitation. Rabbit antisera to rat type IV collagen was raised by intradermal injection of 1 mg purified type IV collagen mixed with complete Freund’s adjuvant. Type IV collagen secreted from and associated with the cells was measured by enzyme-linked immunosorbent assay reported by Rennard et al.5 with slight modification.6 199
200
HANEDA ET AL.
RESULTS AND DISCUSSION Mesangial cells cultured under this experimental condition (containing 20% FCS) reached confluency by day 8 and there was no significant difference in cellular protein and DNA contents among three groups throughout the study period. As shown in Table 1, mesangial cells secreted three to five times more type IV collagen than that associated with the cells. Type IV collagen increased with cell growth in the early log phase (l-6 days) and decreased in late log phase and after confluency (7-12 days). Mesangial cells cultured under high concentrations of glucose secreted a significantly greater amount of type IV collagen than the cells of control or mannitol group (Figure l), resulting in significantly greater accumulation of type IV collagen in the cells under high-glucose conditions. Thus, total accumulation of type IV collagen (by day 12) in control, mannitol, and high-glucose groups was392.1 k 46.1,414.3 k 45.2, and 533.6 k 30.2 kg/mg protein (mean ? SD, n = 4), respectively. These results clearly indicate that glucose is able to enhance the accumulation of type IV collagen in cultured mesangial cells. Although the present study cannot differentiate an increase in biosynthesis from a decrease in degradation of type IV collagen, recent reports in other types of cells7 favor the former hypothesis. Since we have found that glucose does induce metabolic derangements such as an increase in the activity of polyol pathway and a decrease in myoinositol content in cultured mesangial cells,3e4 this metabolic derangement might mediate the effects of glucose on type IV collagen production. Because mesangial expansion has been considered a major abnormality of diabetic glomeruli, enhancement of type IV collagen production in glomerular mesangial cells by glucose could have a significant implication on the pathogenesis of diabetic nephropathy.
TABLE
1
Days Secreted Cellassociated
Type IV Collagen Secreted from and Associated with Cultured Mesangial Cells l-4
697
5, 6
9-12
26.5 2 10.2 50.4 2 4.7 26.2 k 4.7 19.4 ” 1.2 7.2 2 1.8
Note: Flmg protlday,
18.4 + 2.6
mean
f
5.5 ? 2.8
SD, n = 4.
3.3 + 2.5
i
l-4 (30)
5.6
9-12 &3:
(24)
(12)
Days FIG. 1 Type IV collagen secreted from glomerular mesangial cells cultured under 5.6 mM glucose (control, o), 5.6 mM glucose plus 22.2 mM mannitol (mannitol, o), or 27.8 mM glucose (b) conditions. Values shown are kg/mg protein/day (mean 2 SD). Numbers are shown in parentheses. l P < .O? vs. control and mannitol.
REFERENCES 1. Mauer SM, Steffes MW, Ellis EN, Sutherland DER, Brown DM, Goetz FC: Structural-functional relationship in diabetic nephropathy. J C/in Invest 74:1143-1155, 1984. 2. Haralson MA, Jacobson HR, Hoover RL: Collagen polymorphism in cultured rat kidney mesangial cells. Lab invest 57:513-523, 1987. 3. Kikkawa R, Umemura K, Haneda M, Arimura T, Ebata K, Shigeta Y: Evidence for existence of polyol pathway in cultured rat mesangial cells. Diabetes 36:240-243, 1987. 4. Haneda M, Kikkawa R, ArimuraT, Ebata K, Togawa M, Maeda S, Sawada T, Horide N, Shigeta Y: Glucose inhibits myoinositol uptake and reduces myo-inositol content in cultured rat glomerular mesangial cells. Metabolism 39:40-45, 1990. 5. Rennard SI, Berg R, Martin GR, Foidart JM, Robey PG: Enzyme-linked immunoassay (ELISA) for connective tissue components. Anal Biochem 104:205-214, 1980. 6. Haneda M, Kikkawa R, Horide N, Togawa M, Koya D, Kajiwara N, Ooshima A, Shigeta Y: Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 34:198-200, 1991. 7. Cagliero E, Maiello M, Boeri D, Roy S, Lorenzi M: Increased expression of basement membrane components in human endothelial cells cultured in high glucose. J C/in Invest 82:735-738, 1988.
Masakazu Haneda Ryuichi Kikkawa Naoki Horide Masaki Togawa Daisuke Koya Nobuyuki Kajiwara Shiro Maeda Yukio Shigeta Third Department of Medicine, Shiga University of Medical Science, Shiga, Japan
of Type Cells
IV Collagen
ABSTRACT Type IV collagen production by cultured rat glomerular mesangial cells was evaluated quantitatively by measuring type IV collagen secreted into culture media and associated with the cells using enzymelinked immunosorbent assay (ELISA). The majority of type IV collagen was secreted into culture media; type IV collagen increased with cell growth in the early log phase and decreased in the late log phase and after cofluency. By exposing the cells to high concentrations of glucose (27.8 mhl), both secreted and cell-associated type IV collagens increased significantly compared with the cells cultured under normal glucose concentrations (5.6 mM) or under equivalent concentrations of mannitol, resulting in a significant increase in total type IV collagen accumulation. (The Journal of Diabetic Complications 5;23:199-200, 1991.)
INTRODUCTION Expansion of glomerular mesangium has been considered to be one of the major histological characteristics in diabetic nephropathy.’ Glomerular mesangial cells have been reported capable of synthesizing various collagens including type IV collagen2 one of the major components of the mesangial matrix. Therefore, if the production of type IV collagen by mesangial cells is enhanced in diabetes, it could accumulate in the mesangial area, leading to mesangial expansion. In order to prove this hypothesis, we examined the ability of mesangial cells to produce type IV collagen under high-glucose conditions by measuring it quantitatively with enzyme-linked immunosorbent assay (ELISA).
MATERIALSAND METHODS Mesangial Cell Culture Mesangial cells were obtained from the culture of glomeruli isolated from Sprague-Dawley rats as previously described.314 Mesangial cells, plated on a 60-mm culture dish in a density of 77,000 cells per dish, were cultured with RPM1 1640 media containing 20% fetal calf serum (FCS), 50 pg/ml ascorbic acid, and 50 pg/ml paminopropionitrile under the following conditions: (1) 5.6 mM glucose, (2) 27.8 mM glucose, and (3) 5.6 mM glucose plus 22.2 mM mannitol. Media were changed on days 4, 6, 8, and 12, and stored at -20°C until the assay. Cells were washed twice with phosphate-buffered saline (PBS), incubated with PBS containing 0.05% Triton X-100 at 4°C overnight, sonicated, and stored at -20°C. Reprint requests to be sent to: Masakazu Haneda, M.D., Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21, Japan.
0 1991 Elsevier Science Publishing Co., Inc 0891-6632/91/$3,50
Enzyme-Linked lmmunosorbent Assay of Type IV Collagen Type IV COIlagen was isolated from the lungs and kidneys of 20 Sprague-Dawley rats using limited pepsin digestion and differential salt precipitation. Rabbit antisera to rat type IV collagen was raised by intradermal injection of 1 mg purified type IV collagen mixed with complete Freund’s adjuvant. Type IV collagen secreted from and associated with the cells was measured by enzyme-linked immunosorbent assay reported by Rennard et al.5 with slight modification.6 199
200
HANEDA ET AL.
RESULTS AND DISCUSSION Mesangial cells cultured under this experimental condition (containing 20% FCS) reached confluency by day 8 and there was no significant difference in cellular protein and DNA contents among three groups throughout the study period. As shown in Table 1, mesangial cells secreted three to five times more type IV collagen than that associated with the cells. Type IV collagen increased with cell growth in the early log phase (l-6 days) and decreased in late log phase and after confluency (7-12 days). Mesangial cells cultured under high concentrations of glucose secreted a significantly greater amount of type IV collagen than the cells of control or mannitol group (Figure l), resulting in significantly greater accumulation of type IV collagen in the cells under high-glucose conditions. Thus, total accumulation of type IV collagen (by day 12) in control, mannitol, and high-glucose groups was392.1 k 46.1,414.3 k 45.2, and 533.6 k 30.2 kg/mg protein (mean ? SD, n = 4), respectively. These results clearly indicate that glucose is able to enhance the accumulation of type IV collagen in cultured mesangial cells. Although the present study cannot differentiate an increase in biosynthesis from a decrease in degradation of type IV collagen, recent reports in other types of cells7 favor the former hypothesis. Since we have found that glucose does induce metabolic derangements such as an increase in the activity of polyol pathway and a decrease in myoinositol content in cultured mesangial cells,3e4 this metabolic derangement might mediate the effects of glucose on type IV collagen production. Because mesangial expansion has been considered a major abnormality of diabetic glomeruli, enhancement of type IV collagen production in glomerular mesangial cells by glucose could have a significant implication on the pathogenesis of diabetic nephropathy.
TABLE
1
Days Secreted Cellassociated
Type IV Collagen Secreted from and Associated with Cultured Mesangial Cells l-4
697
5, 6
9-12
26.5 2 10.2 50.4 2 4.7 26.2 k 4.7 19.4 ” 1.2 7.2 2 1.8
Note: Flmg protlday,
18.4 + 2.6
mean
f
5.5 ? 2.8
SD, n = 4.
3.3 + 2.5
i
l-4 (30)
5.6
9-12 &3:
(24)
(12)
Days FIG. 1 Type IV collagen secreted from glomerular mesangial cells cultured under 5.6 mM glucose (control, o), 5.6 mM glucose plus 22.2 mM mannitol (mannitol, o), or 27.8 mM glucose (b) conditions. Values shown are kg/mg protein/day (mean 2 SD). Numbers are shown in parentheses. l P < .O? vs. control and mannitol.
REFERENCES 1. Mauer SM, Steffes MW, Ellis EN, Sutherland DER, Brown DM, Goetz FC: Structural-functional relationship in diabetic nephropathy. J C/in Invest 74:1143-1155, 1984. 2. Haralson MA, Jacobson HR, Hoover RL: Collagen polymorphism in cultured rat kidney mesangial cells. Lab invest 57:513-523, 1987. 3. Kikkawa R, Umemura K, Haneda M, Arimura T, Ebata K, Shigeta Y: Evidence for existence of polyol pathway in cultured rat mesangial cells. Diabetes 36:240-243, 1987. 4. Haneda M, Kikkawa R, ArimuraT, Ebata K, Togawa M, Maeda S, Sawada T, Horide N, Shigeta Y: Glucose inhibits myoinositol uptake and reduces myo-inositol content in cultured rat glomerular mesangial cells. Metabolism 39:40-45, 1990. 5. Rennard SI, Berg R, Martin GR, Foidart JM, Robey PG: Enzyme-linked immunoassay (ELISA) for connective tissue components. Anal Biochem 104:205-214, 1980. 6. Haneda M, Kikkawa R, Horide N, Togawa M, Koya D, Kajiwara N, Ooshima A, Shigeta Y: Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 34:198-200, 1991. 7. Cagliero E, Maiello M, Boeri D, Roy S, Lorenzi M: Increased expression of basement membrane components in human endothelial cells cultured in high glucose. J C/in Invest 82:735-738, 1988.