Effekten av hyperglykemi og metylglyoksal på syntesen av proteoglykaner i epitelceller fra distale tubuli in vitro

Abstract

Background: Diabetic nephropathy poses an increasing health problem in the Western world, and research to new leads for diagnosis and therapy is warranted. Morphologically, diabetes nephropathy is characterized by thickening of the glomerular basement membrane (GBM), expansion of the mesangial matrix and podocyte loss. In addition to the GBM, the tubular basement membrane (TBM) also shows thickening, which is already evident at the silent stage of the disease. Long-term exposure to high levels of blood glucose leads to the formation of advanced glycated end products (AGEs). Levels of AGEs correlate with the degree of diabetes nephropathy and both AGEs and hyperglycemia can affect the synthesis and turnover of extracellular matrix components. Changes in kidney function in diabetes could be due to changes in the kidney basement membranes. This study explored the possibility that AGEs and hyperglycemia affect proteoglycan synthesis in cultured epithelial cells from distal tubules. Methods: The Madin Darby Canine Kidney I (MDCK I) cells were cultured on plastic for 2, 4 or 8 days. To mimic the metabolic environment of diabetes in vivo the MDCK I cells were cultured in medium with 25 mM glucose or media with different concentrations of methylglyoxal (MGO) (0.2, 0.5 and 1.0 mM). Medium with low glucose (5 mM) was used as a control and medium with mannitol (5 mM glucose and 20 mM mannitol) was used as a negative osmotic control in all the work with high glucose. The cells were labelled with [35S]-sulphate 24 h before harvesting. The labelled material was purified by Sephadex G-50 fine chromatography and DEAE-ion exchange chromatography, and analysed by SDS-PAGE. The amount of cells, protein and proliferation were determined for all the different conditions after 2, 4 and 8 days. Results: A reduction in the proteoglycan synthesis was induced by 25 mM glucose, in comparison with 5 mM glucose when labelled with [35S]-sulphate. 0.2 and 0.5 mM MGO increased the proteoglycan expression the first two days, but after 4 and 8 days the amount of proteoglycans was decreased. We found a reduced adhesion when culturing MDCK I cells in 1 mM MGO for 4 days. Although all the cells were floating in the medium, they were still viable and attached to the plastic when they were transferred to fresh medium. MGO also induced an increase in protein content and a decrease in cell number, indicating hypertrophy. Conclusion: The changes in expression of [35S]-proteoglycans induced by high glucose and MGO indicates an involvement of hyperglycemia and AGEs in the regulation of synthesis and turnover of proteoglycans.