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American Journal of Pathology, Vol 150, 1101-1111, Copyright © 1997 by American Society for Investigative Pathology
REGULAR ARTICLES |
AO Phillips, R Steadman, K Morrisey and JD Williams
Institute of Nephrology, University of Wales College of Medicine, Cardiff Royal Infirmary, United Kingdom.
Proximal tubular epithelial cells are the most abundant cells in the renal cortex, and recent studies suggest that they may play an important role in initiating pathological changes in renal disease. Transforming growth factor (TGF)-beta 1 has been implicated as a major factor controlling the development and progression of renal fibrosis in numerous diseases, including diabetic nephropathy. We have recently demonstrated that human proximal tubular epithelial cells synthesize and secrete TGF-beta 1 after the sequential addition of both 25 mmol/L D-glucose and platelet-derived growth factor (PDGF). The present study examines the control of this synthesis and in particular the polar requirements of the stimulation and the direction of release of the protein. A proximal tubular cell line (LLC-PK1) was cultured on porous tissue culture inserts. Confluent cells were exposed to 25 mmol/L D- glucose on either their apical or basolateral aspect. TGF-beta 1 mRNA induction (reverse transcriptase polymerase chain reaction) occurred only after basolateral exposure. Similarly, TGF-beta 1 synthesis and secretion was induced only by the subsequent addition of PDGF to the basolateral aspect of the cells. In contrast, TGF-beta 1 protein secretion was detected equally in the apical and basolateral compartments. This effect was maximal after 12-hour PDGF stimulation and represented a threefold increase over controls for TGF-beta 1 in both the apical and basolateral compartments (n = 3, P < 0.05 versus control). The glucose transporter inhibitors phlorizin and phloretin were used to investigate the role of specific D-glucose transport proteins. Application of either basolateral phlorizin or phloretin at the time of addition of 25 mmol/L D-glucose to the same compartment inhibited TGF-beta 1 synthesis in response to PDGF. Maximal inhibition was achieved at 0.5 mmol/L of either inhibitor (phlorizin percent inhibition of apical TGF-beta 1, 75%, P = 0.015, and of basolateral TGF- beta 1, 78%, P = 0.015; phloretin percent inhibition of apical TGF-beta 1, 68%, P = 0.03, and of basolateral TGF-beta 1, 79%, P = 0.001, n = 5, P versus control). No inhibition was seen with apical application of either inhibitor. These data demonstrate that the priming of proximal tubular cells for TGF-beta 1 synthesis occurs only after basolateral exposure of the cells to 25 mmol/L D-glucose. This mechanism is dependent on the activity of the basolateral D-glucose transporter GLUT- 1. In another series of experiments, TGF-beta 1 synthesis in response to the addition of basolateral PDGF was also induced after basolateral pretreatment with D-galactose but not 2-deoxy-D-glucose. This priming effect demonstrates the dependence of this response on glucose metabolism by the cells, not simply the activity of the GLUT-1 transporter, as both 2-deoxy-D-glucose and D-galactose are transported by GLUT-1, although only the latter is metabolized. The extrapolation of these results to diabetic nephropathy would suggest that it is changes in the interstitial concentration of glucose rather than the urinary glucose level that likely modulate the synthesis of the profibrotic cytokine TGF-beta 1 and thereby influence the progression of interstitial fibrosis.
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