Elevated D-glucose concentrations modulate TGF-beta 1 synthesis by human cultured renal proximal tubular cells. The permissive role of platelet-derived growth factor.
Interstitial fibrosis is a marker of progression of renal impairment in diabetic nephropathy. Transforming growth factor (TGF)-beta 1 is one of a group of pro-fibrotic cytokines and growth factors that have been associated with the development of interstitial fibrosis. We have examined the modulating influence of glucose on the production of TGF-beta 1 by cultured human proximal tubular cells. Incubation of growth-arrested human proximal tubular cells (HPTC) (72 hours in serum free medium) in 25 mmol/L D-glucose resulted in increased expression of TGF-beta 1 mRNA (as assessed by reverse transcription polymerase chain reaction). This was apparent after 6 hours and increased up to 120 hours exposure. TGF-beta 1 secretion, however, as measured by specific enzyme-linked immunoassay, was unaffected by exposure to 25 mmol/L D-glucose. Sequential stimulation of HPTC, first with 25 mmol/L D-glucose for 48 hours and then with platelet-derived growth factor (PDGF) isoforms, resulted in a dose-dependent secretion of TGF-beta 1. Pre-exposure to 5 mmol/L D-glucose or 25 mmol/L L-glucose did not prime for TGF-beta 1 release. At 50 ng/ml PDGF this effect was greatest for the AA isoform (AA 31.4 +/- 7.1, AB 20.98 +/- 8.9, BB 7.8 +/- 2.2, P < 0.05 for all versus control, n = 3, mean +/- SEM ng/10(6) cells/24 hours). These effects were blocked by the addition of antibody to the PDGF alpha-receptor. TGF-beta 1 secretion was inhibited in a dose-dependent manner by pretreatment with cyclohexamide, but was not affected by pretreatment with actinomycin D. Stimulation of HPTC with a single dose of PDGF induced TGF-beta 1 mRNA; however, only after application of a second dose of PDGF (after TGF-beta 1 mRNA induction) did TGF-beta 1 protein secretion occur. We also demonstrated that PDGF stimulation of HPTC induced an inherently more stable TGF-beta 1 mRNA transcript. These findings demonstrate that elevated D-glucose concentration alone is insufficient to lead to increased TGF-beta 1 secretion by HPTC despite increased mRNA expression. However, application of a second stimulus such as PDGF, when TGF-beta 1 mRNA expression is increased, leads to increased protein synthesis and secretion of TGF-beta 1. This implies that elevated glucose concentrations might prime proximal tubular cells for TGF-beta 1 synthesis and thus contribute to the development of interstitial fibrosis.
Phillips
A O
AO
Institute of Nephrology, University of Wales College of Medicine, Cardiff Royal Infirmary, United Kingdom.
Steadman
R
R
Topley
N
N
Williams
J D
JD
D001483:Base Sequence; D002478:Cells, Cultured; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D009994:Osmolar Concentration; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics
D015345:Oligonucleotide Probes; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D005947:Glucose