J Korean Med Sci.  2006 Jun;21(3):430-435. 10.3346/jkms.2006.21.3.430.

The Inhibitory Effect of siRNAs on The High Glucose-Induced Overexpression of TGF-beta1 in Mesangial Cells

• Noh HJ
• Kim HC
• Lee SS
• Kang YN
• Chae YM
• Park KK

• KMID: 1778423
• DOI: http://doi.org/10.3346/jkms.2006.21.3.430

Abstract

Diabetic nephropathy is characterized by an expansion of the glomerular mesangium, caused by mesangial cell proliferation and an excessive accumulation of extracellar matrix (ECM) proteins, which eventually leading to glomerulosclerosis. TGF-beta1 was found to play an important role in the accumulation of ECM in the kidney. In this study, TGF-beta1 RNA interference was used as an effective therapeutic strategy. The inhibitory effect of TGF-beta1 small interfering RNAs (siRNAs) on the high glucose-induced overexpression of TGF-beta1 in rat mesangial ceys (RMCs). A high levels of glucose induces TGF-beta1 mRNA and protein, and TGF-beta1 siRNAs reduce the ability of high glucose to stimulate their expression. We also examined the inhibitory effect of TGF-beta1 siRNAs on the expression of plasminogen activator inhibitor (PAI)-1 and Collagen Type I which are down-regulators of TGF-beta1. The expression of TGF-beta1, PAI-1 and Collagen Type I was increased in RMCs that were stimulated by 30 mM glucose. TGF-beta1 siRNAs reduces high glucose-induced TGF-beta1, PAI-1, and Collagen Type I mRNA and protein expression in a dose-dependent manner. In conclusion, the present study demonstrates that TGF-beta1 siRNAs effectively inhibits TGF-beta1 mRNA and protein expression in RMCs. These suggest that TGF-beta1 siRNAs through RNAi may be a useful tool for developing new therapeutic applications for the treatment of diabetic nephropathy.

Keyword

Transforming Growth Factors; Transforming Growth Factor beta; Plasminogen Inactivators; Plasminogen Activator Inhibitor 1; Collagen Type I; RNA, Small Interfering

MeSH Terms

Transforming Growth Factor beta1/*metabolism
Rats, Sprague-Dawley
Rats
RNA, Small Interfering/*metabolism
Microscopy, Fluorescence
Mesangial Cells/*metabolism
Male
Glucose/*metabolism
Glomerular Mesangium/*metabolism
Gene Expression Regulation
Diabetic Nephropathies/pathology
Collagen Type I/metabolism
Cells, Cultured
Cell Proliferation
Animals

Figure

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  Fig. 1 TGF-β1 stimulated by 5.5, 20, 30 and 40 mM glucose in RMCs. After incubation of RMCs with control (5.5 mM), high (20 mM), high (30 mM), high (40 mM) glucose for 24 hr, total RNA was isolated, reverse transcripted and amplified by the polymerase chain reaction (A). TGF-β1 levels for each conditions were measured by means of an LAS-3000 image analyzer (B). The expression level of TGF-β1 increases in 30 mM high glucose.

• 

  Fig. 2 Fluorescence microscopy photograph of RMCs transfected with TGF-β1 siRNAs in cationic liposomes. The fluorescein labeled TGF-β1 siRNAs are transfected to RMCs (×200).

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  Fig. 3 The effect of TGF-β1 siRNAs on high glucose induced TGF-β1, PAI-1 and Collagen Type I protein upregulation in RMCs (Western blot). (A) Lane 1: control (5.5 mM), Lane 2: stimulated with high glucose (30 mM), Lane 3: 2 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. Lane 4: 4µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. Lane 5: 8 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. (B) Values are presented as means ±SEM of triplicate experiments.*p<0.05 compared to normal glucose;†p<0.05 compared to high glucose.

• 

  Fig. 4 The effect of TGF-β1 siRNAs on the high glucose-induced mRNA expression of TGF-β1, PAI-1 and Collagen Type I. RMCs transfected with TGF-β1 siRNAs were cultured in medium containing 5.5 or 30 mM glucose for 24 hr and total RNA was extracted from the RMCs. mRNA expression of TGF-β1, PAI-1 and Collagen Type I was determined by RT-PCR (A). TGF-β1, PAI-1 and Collagen Type I levels for each condition was measured by means of an LAS-3000 image analyzer (B). Lane 1: control (5.5 mM), Lane 2: stimulated with high glucose (30 mM), Lane 3: 1 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. Lane 4: 2 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. Lane 5: 4 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. Values are presented as means±SEM of triplicate experiments. *p<0.05 compared to normal glucose;†p<0.05 compared to high glucose.

• 

  Fig. 5 TGF-β1 siRNAs suppress high glucose-induced RMC proliferation. RMCs transfected with TGF-β1 siRNAs were cultured in medium containing normal glucose or high glucose for 24 hr, and the proliferation was determined by the WST-1 test. N: control (5.5 mM), H: stimulated with high glucose (30 mM), 0.5X: 0.1µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. 1X: 0.2 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulation for 24 hr. 2X: 0.4 µg/µL TGF-β1 siRNAs transfection plus high glucose (30 mM) stimulationfor 24 hr. Values are presented as means±SEM of triplicate experiments. *p<0.05 compared to normal glucose;†p<0.05 compared to high glucose.

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