Expression of Endothelin-1 and Its Receptors in Cisplatin-Induced Acute Renal Failure in Mice

Seokwoo Lee; Dowhan Ahn

doi:10.4196/kjpp.2008.12.4.149

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Lee and Ahn: Expression of Endothelin-1 and Its Receptors in Cisplatin-Induced Acute Renal Failure in Mice

Original Article

Korean J Physiol Pharmacol 2008;12(4):149-153.

Published online: 17 August 2008

DOI: https://doi.org/10.4196/kjpp.2008.12.4.149

Expression of Endothelin-1 and Its Receptors in Cisplatin-Induced Acute Renal Failure in Mice

Seokwoo Lee, Dowhan Ahn

Department of Physiology, Kosin University College of Medicine, Busan 602-703, Korea

Corresponding to: Dowhan Ahn, Department of Physiology, Kosin University College of Medicine, 35, Amnam-dong, Seo-gu, Busan 602-703, Korea. (Tel) 82-51-990-6415, (Fax) 82-51-990-3081, (E-mail) dwahn@kosin.ac.kr

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Endothelin-1 (ET-1) is unequivocally elevated in the kidney with ischemic acute renal failure (ARF), whereas ET receptors (ET_AR and ET_BR) are variably expressed. Although renal functional and structural changes are similar between ischemic and nephrotoxic ARF, there are few reports on the alteration in the ET system in nephrotoxic ARF. This study was, therefore, undertaken to investigate changes in renal expression of ET-1 and its receptors in nephrotoxic ARF induced by cisplatin. Mice were intraperitoneally injected with 16 mg of cisplatin/kg at a single dose, and the expression of mRNA and protein was then quantified by real-time RT-PCR and Western blot, respectively. Immuno-histochemistry was conducted for localization. Three days after treatment, ET-1 transcript in cisplatintreated mice was thirteen times higher than that in controls, whereas ET-1 peptide was increased by 1.5-fold. Cisplatin caused a 2-fold increase in the levels of ET_AR mRNA and protein. Most of the increased immunoreactive ET-1 and ET_AR were localized in damaged tubules. Neither the expression of ET_BR mRNA nor the abundance and immunoreactive level of ET_BR protein were changed. The findings suggest that the individual components of the renal ET system are differentially regulated in cisplatin-induced nephrotoxic ARF.

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Keywords: Cisplatin, Acute renal failure, Endothelin, Endothelin receptor

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Fig. 1.

Relative expression of ET-1, ET_AR, and ET_BR genes in the kidney of the control and cisplatin-treated mice. Real-time RT-PCR was performed as described in Materials and Methods. Relative expression was calculated by a comparative C_T method. Values are mean±SD of 3 separate experiments. ∗p<0.05 vs. matched control.

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Fig. 2.

ET-1 contents in kidney homogenates of the control and cisplatin-treated mice. Values are mean±SD of 6 animals. ∗p<0.05 vs. control.

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Fig. 3.

Representative immunoblots (upper panel) and densitometric analysis (lower panel) of ET-1, ET_AR, and ET_BR proteins. Each blot was loaded with 50 ug of protein. Band density was normalized to β-actin. Values are mean±SD of 3 separate experiments. ∗p<0.05 vs. respective control.

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Fig. 4.

Immunostaining for ET-1, ET_AR, and ET_BR in the renal cortex of control and cisplatin-treated mice. ET-1, ET_AR, and ET_BR are diffusely expressed in tubular segments. Note that intense immuno-staining for ET-1 and ET_AR is found in the cisplatin- treated group. Magnification: ×100.

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Table 1.

Primer sequences for real time RT-PCR

Gene	Sequence	Size	GenBank
ET-1	F 5'-ACT TCT GCC ACC TGG GCA TC-3'	199	NM 010104
	R 5'-ACT TTG GGC CCT GAG TTC TT-3'
ET_AR	F 5'-ACG GTC TTG AAC CTC TGT GC −3'	263	NM 010332
	R 5'-AGC CAC CAG TCC TTC ACA TC −3'
ET_BR	F 5'-AGC TGG TGC CCT TCA TAC AG-3'	231	NM 007904
	R 5'-GGG GCT TTC CTT TGT AGT CC-3'
β-Actin	F 5'-GAC GGC CAG GTC ATC ACT AT-3'	216	NM 007393
	R 5'-CTT CTG CAT CCT GTC AGC AA-3'

ET-1, endothelin-1; ET_AR, endothelin A receptor; ET_BR, endothelin B receptor.

Table 2.

Changes in BUN and plasma creatinine concentration

	Control	Cisplatin
BUN (mg/dl)	18.3±4.0	107±31.6^∗
Pcreatinine (mg/dl)	0.72±0.07	1.11±0.16^∗

Blood was obtained 72-hr after saline or cisplatin (16 mg/kg) injection. Values are mean±SD of 6 animals.

^∗ p<0.05 vs. control

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