Epigenetic and genetic analysis of p16 in dermal fibroblasts from type 1 diabetic patients with nephropathy.
BACKGROUND
Several studies have shown that cultured skin fibroblasts from patients with diabetic nephropathy (DN) exhibit a hyperplastic growth phenotype. Increased DNA synthesis in cells from patients with DN may ultimately involve alterations in cell cycle regulatory proteins. p16 protein is a member of INK4 family of cyclin-dependent kinase inhibitors, which plays an important role in cell cycle regulation. In this study, we examined the correlation between p16 protein expression in cultured dermal fibroblasts from type 1 diabetic patients and the presence of DN.


METHODS
Western blot analysis was performed to compare p16 protein expression in skin fibroblasts from patients with DN as compared to control subjects, diabetic patients without DN, and nondiabetic patients with nephropathy. Transcriptional regulation of the p16 gene was assessed using competitive reverse transcription-polymerase chain reaction (RT-PCR). Methylation status of the promoter region of p16 was examined using methylation-specific PCR, and we used single-stranded conformational polymorphism (SSCP)-PCR to assess p16 single-nucleotide polymorphism.


RESULTS
Cells from diabetic patients with DN had nondetectable to significantly lower protein expression of p16. Similarly, mRNA expression of p16 was significantly lower in diabetic patients with DN. No hypermethylation of p16 gene was detected, and no abnormal migrating bands were noticed on SSCP-PCR analysis in cells from patients with DN.


CONCLUSIONS
Our data indicate that cells from patients with DN exhibit significantly lower protein and mRNA expression of p16. This study could have not only important implications for the understanding of the pathogenesis of DN, but also the absence of p16 may ultimately serve as an early marker for DN.
Zeng
Lixia
L
Department of Medicine, Division of Nephrology/Hypertension, The Feinberg School of Medicine of Northwestern University, Chicago, Illinois 60611, USA.
Kanwar
Yashpal S
YS
Amro
Nail
N
Phillips
Carrie
C
Molitch
Mark
M
Batlle
Daniel
D
Danesh
Farhad R
FR
D000328:Adult; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D020405:Dermis / Q000166:cytology; D003922:Diabetes Mellitus, Type 1 / Q000503:physiopathology*; D003928:Diabetic Nephropathies / Q000503:physiopathology*; D044127:Epigenesis, Genetic; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis
D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger