Effect of Fenoldopam on Ischemia/Reperfusion-Induced Apoptosis

Figures & data

Table 1 Primer sequences and annealing conditions for polymerase chain reaction

Gene	Primer sequences	Product size	PCR conditions
Bax	Forward		Denaturation 94°C for 50 sec
	5′-CTGCAGAGGATGATTGCTGA-3′	174 bp	Annealing 61°C for 50 sec
	Reverse		Extension 72°C for 1 min
	5′-GATCAGCTCGGGCACTTTAG-3′		Number of cycles: 30
Bad	Forward		Denaturation 94°C for 50 sec
	5′-CAGTGATCTGCTCCACATTC-3′		Annealing 61°C for 50 sec
	Reverse	340 bp	Extension 72°C for 1 min
	5′-TCCAGCTAGGATGATAGGAC-3′		Number of cycles: 30
Bclx	Forward		Denaturation 94°C for 50 sec
	5′-AGGATACAGCTGGAGTCAG-3′		Annealing 61°C for 50 sec
	Reverse	417 bp	Extension 72°C for 1 min
	5′-TCTCCTTGTCTACGCTTTCC-3′		Number of cycles: 30
Bcl2	Forward		Denaturation 94°C for 50 sec
	5′-GCTACGAGTGGGATACTGG-3′		Annealing 61°C for 50 sec
	Reverse	472 bp	Extension 72°C for 1 min
	5′-GTGTGCAGATGCCGGTTCA-3′		Number of cycles: 30
Gapdh	Forward		Denaturation 94°C for 1 min
	5′-GGT CGG TGT GAA CGG ATT TG-3′		Annealing 60°C for 1 min
	Reverse	350 bp	Extension 72°C for 1 min
	5′-GTG AGC CCC AGC CTT CTC CAT-3′		Number of cycles: 30

Figure 1 Effect of fenoldopam on ischemia/reperfusion induced apoptosis. Quantitative measurement of DNA fragmentation in nuclei of renal (A) cortex and (B) medulla. Each tissue section was analyzed in at least 20 areas (10 in cortex and 10 in medulla) by two blinded investigators, and the mean number of stained nuclei was calculated. The original magnification for nuclei counting was ×200. Group-wise differences were calculated using ANOVA.

Figure 2 (A) Effect of fenoldopam on ischemia/reperfusion-induced apoptosis signal transduction genes. All 73 genes up-regulated (stringent criteria ≥ two-fold) by I/R injury were returned to the baseline level or less with fenoldopam. In each group, total RNA from six individual animals were pooled and subjected to microarray analysis. Each spot in the figure represents the expression of the particular gene in each group. A connecting line is drawn for every single gene between groups to illustrate the level of induction and attenuation for each gene after I/R injury with saline and fenoldopam, respectively. (B) RT-PCR analysis of Bax, Bad, Bcl2, and Bclx mRNA expression in rat kidney after sham operation and ischemia/reperfusion with or without fenoldopam. GAPDH was used as positive control. Negative controls without template RNA were shown in lane 1. (C) Correlation analysis of the expression levels for Bax, Bad, Bcl2, and Bclx between the microarray analysis and RT-PCR analysis.