J Korean Med Sci.  2004 Jun;19(3):374-383. 10.3346/jkms.2004.19.3.374.

Effect of Antisense TGF-beta1 Oligodeoxynucleotides in Streptozotocin-Induced Diabetic Rat Kidney

• Jeong HS
• Park KK
• Kim SP
• Choi IJ
• Lee IK
• Kim HC

• KMID: 1786815
• DOI: http://doi.org/10.3346/jkms.2004.19.3.374

Abstract

Transforming growth factor (TGF)-beta1 is an important fibrogenic factor that is involved in the pathogenesis of diabetic nephropathy. We evaluated the effect of circular antisense TGF-beta1 oligodeoxynucleotides (ODNs) on the TGF-beta1 expression in the rat mesangial cell culture and in streptozotocin (STZ)-induced diabetic rats. Circular antisense TGF-beta1 ODNs were found to be stable in rat serum, significantly decreasing TGF-beta1 mRNA expression compared with linear antisense ODNs in the rat mesangial cell culture. Circular antisense TGF-beta1 ODNs were introduced into the tail vein of normal rats using hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method and were confirmed to be delivered effectively into the kidney, liver, lungs, and spleen. To inhibit the overexpression of TGF-beta1 in diabetic kidneys, we introduced circular antisense TGF-beta1 ODNs into the STZ-induced diabetic rats. On day 13 after circular antisense TGF-beta1 ODNs injection, TGF-beta1 mRNA and protein expression markedly decreased and urinary TGF-beta1 excretion rate also dropped in the circular antisense TGF-beta1 ODNs-treated diabetic rats. These results suggest that circular antisense TGF-beta1 ODNs may be a useful tool for developing new therapeutic application for progressive diabetic nephropathy.

Keyword

Transforming Growth Factors; Diabetes Mellitus; Oligonucleotides; Streptozotocin

MeSH Terms

Animals
Blood Glucose/metabolism
Cells, Cultured
Diabetes Mellitus, Experimental/*therapy
Enzyme-Linked Immunosorbent Assay
Gene Transfer Techniques
Immunohistochemistry
Liposomes/chemistry
Male
Microscopy, Confocal
Oligonucleotides, Antisense/metabolism/pharmacology/*therapeutic use
RNA/metabolism
RNA, Messenger/metabolism
Rats
Rats, Sprague-Dawley
Reverse Transcriptase Polymerase Chain Reaction
Streptozocin
Support, Non-U.S. Gov't
Time Factors
Transfection
Transforming Growth Factor beta/*genetics

Figure

• 

  Fig. 1 Photograph of stained 6% denaturing polyacrylamide gel showing relative mobilities of linear and circular antisense TGF-β1 oligodeoxynucleotides (ODNs). (A) Synthesis of circular antisense TGF-β1 ODNs from linear ODNs: lane 1, 58-nt linear antisense TGF-β1 ODNs; lane 2, 116-nt circular antisense TGF-β1 ODNs. (B) Stability of circular and linear antisense TGF-β1 ODNs after the treatment with rat serum: lane 1, untreated circular antisense TGF-β1 ODNs; lane 2, circular antisense TGF-β1 ODNs treated with 50% (v/v) rat serum for 24 hr; lane 3, untreated linear antisense TGF-β1 ODNs; lane 4, linear antisense TGF-β1 ODNs treated with 50% (v/v) rat serum for 24 hr.

• 

  Fig. 2 Fluorescence microscopy of rat mesangial cells transfected with circular antisense TGF-β1 oligodeoxynucleotides (ODNs) in cationic liposomes. Fluorescein-labeled-circular antisense TGF-β1 ODNs were observed in both the nuclei and the cytoplasm (×400).

• 

  Fig. 3 Effect of circular antisense oligodeoxynucleotides (ODNs) on TGF-β1 mRNA expression in rat mesangial cells (RMCs). RMCs were serum-starved for 48 hr and subsequently transfected with circular antisense ODNs (1 µg)+cationic liposome (4 µg) (lane 1), circular antisense ODNs (0.5 µg)+cationic liposome (4 µg) (lane 2), linear antisense ODNs (1 µg)+cationic liposome (4 µg) (lane 3), linear antisense ODNs (0.5 µg)+cationic liposome (4 µg) (lane 4), circular sense ODNs (1 µg)+cationic liposome (4 µg) (lane 5), and cationic liposome (4 µg) alone (lane 6), and then exposed to RPMI 1640 containing 17% FCS. Total RNA was isolated from the RMCs and subjected to RT-PCR (TGF-β1: 598 bp, GAPDH: 351 bp).

• 

  Fig. 4 Confocal microscopy of rat organs transfected with fluorescein-labeled-circular antisense TGF-β1 ODNs in HVJ-liposome by intravenous systemic administration method (A: kidney, B: liver, C: lung, D: spleen, ×200).

• 

  Fig. 5 RT-PCR analysis of the effect of circular antisense oligodeoxynucleotides (ODNs) on TGF-β1 mRNA expression in diabetic kidneys on days 1, 5, and 13 after injection of ODNs into diabetic rats. Lane 1, normal kidney; lane 2, on day 1, untreated diabetic kidney; lane 3, on day 1, ODNs-treated diabetic kidney; lane 4, on day 5, untreated diabetic kidney; lane 5, on day 5, ODNs-treated diabetic kidney; lane 6, on day 13, untreated diabetic kidney; lane 7, on day 13, ODNs-treated diabetic kidney (TGF-β1: 598 bp, GAPDH: 351 bp).

• 

  Fig. 6 Immunohistochemical staining of TGF-β1. On day 1 after injection of circular antisense TGF-β1 oligodeoxynucleotides (ODNs) into diabetic rats, ODNs-treated diabetic kidney (B), on day 5, ODNs-treated diabetic kidney (D), on day 13, ODNs-treated diabetic kidney (G), and their respective controls (A, C, E, F: untreated diabetic kidney) (×200).

• 

  Fig. 7 Effect of circular antisense oligodeoxynucleotides (ODNs) on urinary TGF-β1 excretion rate. Collected urine samples on days 1, 5, and 13 after injection of ODNs into diabetic rats were assayed by a TGF-β1 ELISA (normal: open bars, diabetes: hatched bars, ODNs-treated diabetes: solid bars). Data are expressed as mean±SE. *: p<0.05 (normal vs. diabetes).

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