Journal List > Korean J Physiol Pharmacol > v.12(4) > 1025544

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

Abstract

Endothelin-1 (ET-1) is unequivocally elevated in the kidney with ischemic acute renal failure (ARF), whereas ET receptors (ETAR and ETBR) 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 ETAR mRNA and protein. Most of the increased immunoreactive ET-1 and ETAR were localized in damaged tubules. Neither the expression of ETBR mRNA nor the abundance and immunoreactive level of ETBR 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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Fig. 1.
Relative expression of ET-1, ETAR, and ETBR 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 CT method. Values are mean±SD of 3 separate experiments. ∗p<0.05 vs. matched control.
kjpp-12-149f1.tif
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.
kjpp-12-149f2.tif
Fig. 3.
Representative immunoblots (upper panel) and densitometric analysis (lower panel) of ET-1, ETAR, and ETBR 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.
kjpp-12-149f3.tif
Fig. 4.
Immunostaining for ET-1, ETAR, and ETBR in the renal cortex of control and cisplatin-treated mice. ET-1, ETAR, and ETBR are diffusely expressed in tubular segments. Note that intense immuno-staining for ET-1 and ETAR is found in the cisplatin- treated group. Magnification: ×100.
kjpp-12-149f4.tif
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'    
ETAR F 5'-ACG GTC TTG AAC CTC TGT GC −3' 263 NM 010332
  R 5'-AGC CAC CAG TCC TTC ACA TC −3'    
ETBR 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; ETAR, endothelin A receptor; ETBR, 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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