J Korean Med Sci.  2009 Feb;24(1):138-145. 10.3346/jkms.2009.24.1.138.

Endothelin A Receptor Blockade Influences Apoptosis and Cellular Proliferation in the Developing Rat Kidney

• Yoo KH
• Yim HE
• Jang GY
• Bae IS
• Choi BM
• Hong YS
• Lee JW

• KMID: 1794420
• DOI: http://doi.org/10.3346/jkms.2009.24.1.138

Abstract

Endothelin systems are believed to play important roles in the emergence and maintenance of functions of various organs during perinatal development, including the kidney. The present study was designed to investigate the roles of endothelin systems on physiologic renal growth and development. Newborn rat pups were treated with either Bristol-Myers Squibb-182874 (30 mg/kg/day), a selective endothelin A receptor (ET(A)R) antagonist, or vehicle for 7 days. To identify cellular changes, kidneys were examined for apoptotic cells by terminal deoxynucleotide transferasemediated nick-end labeling stain and proliferating cell nuclear antigen (PCNA) by immunohistochemical (IHC) stain. To clarify the molecular control of these processes, immunoblots and reverse transcriptase-polymerase chain reaction for Clusterin, Bcl-2, Bcl-X(L), Bax, and p53 were performed. ETAR antagonist treatment resulted in reduced kidney weights, decreased PCNA-positive proliferating cells, and increased apoptotic cells. The protein expressions of renal Bcl-X(L) and Bax in the ETAR antagonist-treated group were significantly decreased, whereas the mRNA expressions of these genes were not changed. There were no significant differences in the expressions of Clusterin, Bcl-2, and p53. In conclusion, inhibition of endogenous endothelins impairs renal growth, in which decreased cellular proliferation, increased apoptosis and decreased expressions of renal Bcl-X(L) and Bax are possibly implicated.

Keyword

Endothelins; Growth and Development; Apoptosis; Cell Proliferation; Clusterin; Bcl-2; Bcl-XL; Bax; p53; Kidney

MeSH Terms

Animals
Animals, Newborn
Antihypertensive Agents/*pharmacology
*Apoptosis
*Cell Proliferation
Dansyl Compounds/*pharmacology
Gene Expression Regulation, Developmental/drug effects
In Situ Nick-End Labeling
Kidney/drug effects/*growth & development/*metabolism
Proliferating Cell Nuclear Antigen/metabolism
Proto-Oncogene Proteins c-bcl-2/genetics/metabolism
Rats
Rats, Sprague-Dawley
Receptor, Endothelin A/*antagonists & inhibitors/metabolism
Reverse Transcriptase Polymerase Chain Reaction
Tumor Suppressor Protein p53/genetics/metabolism
bcl-X Protein/genetics/metabolism

Figure

• 

  Fig. 1 PCNA stains for proliferating cells in the control group (A) and the BMS group (B). PCNA-positive cells were rarely found in the BMS group as compared with the control group. Cortical tubular epithelial and interstitial cell proliferations were decreased following treatment with the ETA receptor antagonist as compared with the control group. Bar+0.1 µm. Magnification: ×400.

• 

  Fig. 2 TUNEL stains for apoptotic cells in the control group (A) and the BMS group (B). In the BMS group, TUNEL-positive apoptotic cells were more frequently found in the dilated tubular epithelial cells, as compared with the control group. Bar+0.1 µm. Magnification: ×400.

• 

  Fig. 3 Representative expressions of clusterin mRNA and protein. ▓, control group; ▒, BMS group. (A) On the semi-quantitative RT-PCR, the clusterin/GAPDH mRNA expressions were not different between the two groups. (B) On the immunoblot analysis, the clusterin/tubulin protein expressions were not changed in the BMS group as compared with the control group.

• 

  Fig. 4 Representative expressions of Bcl-2 mRNA and protein. ▓, control group; ▒, BMS group. (A) On the semi-quantitative RT-PCR, the Bcl-2/GAPDH mRNA expression was not different between the two groups. (B) On the immunoblot analysis, the Bcl-2/tubulin protein expression were not changed in the BMS group as compared with the control group.

• 

  Fig. 5 Representative expression of Bcl-XL mRNA and protein. ▓, control group; ▒, BMS group. (A) On the semi-quantitative RT-PCR, the Bcl-XL/GAPDH mRNA expression were not different between the two groups. (B) On the immunoblot analysis, the Bcl-XL/tubulin protein expression was decreased significantly in the BMS group as compared with the control group.

• 

  Fig. 6 Representative expression of Bax mRNA and protein. ▓, control group; ▒, BMS group. (A) On the semi-quantitative RT-PCR, the Bax/GAPDH mRNA expression were not different between the two groups. (B) On the immunoblot analysis, the Bax/tubulin protein expression was decreased significantly in the BMS group as compared with the control group.

• 

  Fig. 7 Representative expression of p53 mRNA and protein. ▓, control group; ▒, BMS group. (A) On the semi-quantitative RT-PCR, the p53/GAPDH mRNA expression was not different between the two groups. (B) On the immunoblot analysis, the p53/tubulin protein expression was not changed in the BMS group as compared with the control group.

Reference

1. Uchide T, Fujimori Y, Sasaki T, Temma K, Adur J, Masuo Y, Kozakai T, Lee YS, Saida K. Expression of endothelin-1 and vasoactive intestinal contractor genes in mouse organs during the perinatal period. Clin Sci (Lond). 2002. 103:Suppl 48. 167S–170S.

2. Chan TS, Lin CX, Chan WY, Chung SS, Chung SK. Mouse preproendothelin-1 gene. cDNA cloning, sequence analysis and determination of sites of expression during embryonic development. Eur J Biochem. 1995. 234:819–826.

3. Adur J, Takizawa S, Quan J, Uchide T, Saida K. Increased gene expression and production of murine endothelin receptors after birth. Biochem Biophys Res Commun. 2003. 305:700–706.

4. Kurihara Y, Kurihara H, Oda H, Maemura K, Nagai R, Ishikawa T, Yazaki Y. Aortic arch malformations and ventricular septal defect in mice deficient in endothelin-1. J Clin Invest. 1995. 96:293–300.

5. Clouthier DE, Hosoda K, Richardson JA, Williams SC, Yanagisawa H, Kuwaki T, Kumada M, Hammer RE, Yanagisawa M. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998. 125:813–824.

6. Choi BM, Yoo KH, Hong YS, Lee JW, Kim SK. Effects of endothelin A receptor antagonist to cellular proliferation and apoptosis in neonatal rat heart. J Korean Pediatr Soc. 2003. 46:668–678.

7. Choi JH, Yoo KH, Cheon HW, Kim KB, Hong YS, Lee JW, Kim SK, Kim CH. Angiotensin converting enzyme inhibition decreases cell turnover in the neonatal rat heart. Pediatr Res. 2002. 52:325–332.

8. Choi BM, Yoo KH, Bae IS, Oh MH, Hong YS, Lee JW, Kim SK. Angiotensin-converting enzyme inhibition modulates mitogen-activated protein kinase family expressions in the neonatal rat kidney. Pediatr Res. 2005. 57:115–123.

9. Rossi GP, Sacchetto A, Cesari M, Pessina AC. Interactions between endothelin-1 and the renin-angiotensin-aldosterone system. Cardiovasc Res. 1999. 43:300–307.

10. Gomez-Garre D, Ruiz-Ortega M, Ortego M, Largo R, Lopez-Armada MJ, Plaza JJ, Gonzalez E, Egido J. Effects and interactions of endothelin-1 and angiotensin II on matrix protein expression and synthesis and mesangial cell growth. Hypertension. 1996. 27:885–892.

11. Kusserow H, Unger T. Vasoactive peptides, their receptors and drug development. Basic Clin Pharmacol Toxicol. 2004. 94:5–12.

12. Yoo KH, Wolstenholme JT, Chevalier RL. Angiotensin-converting enzyme inhibition decreases growth factor expression in the neonatal rat kidney. Pediatr Res. 1997. 42:588–592.

13. Yim HE, Kim MK, Bae IS, Kim JH, Choi BM, Yoo KH, Hong YS, Lee JW. AT1 antagonist modulates activin-like kinase 5 and TGF-beta receptor II in the developing kidney. Pediatr Nephrol. 2006. 21:1377–1388.

14. Yoo KH, Cheon H, Choi BM, Hong YS, Lee JW, Kim SK. Effects of Angiotensin converting enzyme inhibition into apoptosis and related genes in the neonatal rat kidney. J Korean Pediatr Soc. 1999. 42:1086–1095.

15. Kang NS, Yim HE, Bae IS, Choi JH, Choi BM, Yoo KH, Hong YS, Lee JW, Kim SK. ACE inhibition modulates transforming growth factor-beta receptors in the young rat. Pediatr Nephrol. 2003. 18:865–871.

16. Shanmugam S, Corvol P, Gasc JM. Angiotensin II type 2 receptor mRNA expression in the developing cardiopulmonary system of the rat. Hypertension. 1996. 28:91–97.

17. Sorenson CM. Bcl-2 family members and disease. Biochim Biophys Acta. 2004. 1644:169–177.

18. Novack DV, Korsmeyer SJ. Bcl-2 protein expression during murine development. Am J Pathol. 1994. 145:61–73.

19. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993. 74:597–608.

20. Gonzalez-Garcia M, Perez-Ballestero R, Ding L, Duan L, Boise LH, Thompson CB, Nunez G. bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development. 1994. 120:3033–3042.

21. Basu A, Haldar S. The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death. Mol Hum Reprod. 1998. 4:1099–1109.