alpha-MSH decreases apoptosis in ischaemic acute renal failure in rats: possible mechanism of this beneficial effect

Background. Apoptosis frequently occurs in acute renal injury but the molec ular mechanisms responsible for this distinct form of cell death are largel y unknown. Fas belongs to the tumour necrosis factor (TNF)/nerve growth fac tor superfamily and engagement by Fas ligand induces apoptosis in various e pithelial cells. To investigate the role of apoptosis and associated mechan isms, we examined the occurrence of apoptosis and Fas and Fas ligand expres sion, and the therapeutic effect of alpha -melanocyte-stimulating hormone ( alpha -MSH), a potent anti-inflammatory cytokine in an ischaemic acute rena l failure (ARF) rat model. We also examined neutrophil infiltration togethe r with intercellular adhesion molecule-1 (ICAM-1) expression because of the ir possible involvement in apoptosis due to their ability to release variou s inflammatory cytokines and reactive oxygen species. Methods. After unilateral nephrectomy in female Sprague-Dawley rats, the re nal artery of the contralateral kidney was clamped for 40 min and reperfuse d. alpha -MSH or vehicle was injected intraperitoneally immediately after r eperfusion and at 1, 6, or 24 h after reperfusion. The expression of Fas an d Fas ligand was studied by western blot analysis and semiquantitative reve rse transcription polymerase chain reaction (RT-PCR). Apoptosis was assesse d by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-en d labelling (TUNEL) method, and neutrophil infiltration by naphthol AS-D ch loracetate staining. The degree of apoptosis, neutrophil infiltration, and Fas and Fas ligand, and ICAM-1 expression, as well as biochemical and histo logical data were compared between the alpha -MSH and the vehicle-treated g roups. Results. Intraperitoneally administered alpha -MSH significantly reduced re nal injury, measured by blood urea nitrogen (BUN) and creatinine and by the degree of tubular necrosis (109.6 +/- 7.1/54.7 +/- 3.1 mg/dl for BUN, and 1.6 +/- 0.2/1.03 +/- 0.06 mg/dl for creatinine 24 h after ischaemia) (5.4 /- 0.8/2.6 +/- 0.3 for injury score 24 h after ischaemia). Ischaemia caused an increase in Fas and Fas ligand expression and was accompanied by morpho logical evidence of apoptosis. alpha -MSH significantly reduced the degree of apoptosis, as well as Fas and Fas ligand expression (mean apoptotic cell number, 41.7 +/- 31.5/14.2 +/- 2.2 per x 200 field at 24 h after ischaemia . Fas protein expression: sham, 1409 +/- 159 DI (densitometric index); vehi cle/alpha -MSH, 2818 +/- 635/1306 +/- 321 DI at 24 h and 5542 +/- 799/2867 +/- 455 DI at 72 h after ischaemia. Fas ligand protein expression: sham, 12 21 +/- 181 DI; vehicle/alpha -MSH, 2590 +/- 85/1279 +/- 169 DI at 4 h, 4376 +/- 268/2432 +/- 369 DI at 24 h and 5200 +/- 648/ 2253.7 +/- 1104 DI at 72 h after ischaemia). Neutrophil infiltration and ICAM-1 expression were als o significantly reduced in alpha -MSH group (neutrophil infiltration: vehic le/ alpha -MSH, 5.05 +/- 1.8/1.59 +/- 0.4) (ICAM-1 expression, vehicle/alph a -MSH 0.46 +/- 0.21,/0.29 +/- 0.19). Conclusion. These results suggest that apoptosis clearly contributes to tub ular cell loss in ischaemia/ reperfusion (I/R) injury possibly by neutrophi l-mediated pathways or an increase in Fas-Fas ligand expression. The observ ed beneficial effect of alpha -MSH could be related to these mechanisms.