Role of mitogen-activated protein kinase in the regulation of transforming growth factor-beta-induced fibronectin accumulation in cultured renal interstitial fibroblasts.
BACKGROUND
In diabetic nephropathy, tubulointerstitial fibrosis is an important component of renal injury. Transforming growth factor (TGF)-beta is a key cytokine that is involved in the pathogenesis of tubulointerstitial fibrosis. However, signal transduction cascades of TGF-beta under high-glucose conditions remain to be clarified. We undertook this study to elucidate whether mitogen-activated protein (MAP) kinase and Smad proteins were involved in TGF-beta-induced fibronectin (FN) production under high glucose in NRK fibroblasts.


METHODS
After serum restriction, NRK cells were exposed to either normal glucose (5.5 mM d-glucose), high glucose (30 mM d-glucose), or 30 mM l-glucose in the presence or absence of TGF-beta for 24 h. MAP kinase inhibitors (SB 203580 and PD 98059) were added to the cultured NRK fibroblasts 2 h before TGF-beta1, and the incubations continued for 8 h. The phosphorylation of p38 MAP kinase, extracellular signal-regulated kinase (ERK)1/ERK2, and c-Jun N-amino terminal kinase (JNK) was assessed by immunoblotting. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine FN mRNA and protein expression, respectively.


RESULTS
High glucose significantly increased the expression of FN mRNA, by 2.4 +/- 1.4-fold. In the presence of either SB 203580 or PD 98059, the high glucose-induced FN mRNA increase was completely inhibited. Incubation of NRK fibroblasts for 48 h in 30 mM d-glucose did not alter p38 MAP kinase, ERK1/ERK2, or JNK phosphorylation. The addition of exogenous TGF-beta1 (1 ng/ml) for 8 h increased FN mRNA by 2.7 +/- 1.1-fold. Both the TGF-beta1- and high glucose-induced FN mRNA increases were inhibited by SB 203580 and PD 98059. Dominant-negative Smad4 did not affect the FN mRNA increase induced by TGF-beta1 and high glucose. Exogenous TGF-beta1 under both normal and high glucose, enhanced the phosphorylation of both p38 MAP kinase and ERK1/ERK2, but not that of JNK.


CONCLUSIONS
NRK fibroblasts exposed to high glucose demonstrated increased TGF-beta1-induced p38 MAP kinase activation. The FN synthesis induced by high glucose and TGF-beta1 was not affected by the Smads pathway and was not due to increased osmolarity. The enhanced activation of p38 MAP kinase may contribute to the altered fibroblast phenotype that leads to progressive diabetic nephropathy.
Suzuki
Hitoe
H
Department of Medicine, Kidney Center, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
Uchida
Keiko
K
Nitta
Kosaku
K
Nihei
Hiroshi
H
D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051785:Smad Proteins; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005353:Fibronectins; D007093:Imidazoles; D011725:Pyridines; D012333:RNA, Messenger; D051785:Smad Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose; C093642:SB 203580