CHANGES IN EXPRESSION OF SODIUM COTRANSPORTERS AND AQUAPORIN-2 DURING ISCHEMIA-REPERFUSION INJURY IN RABBIT KIDNEY

Figures & data

Table

Name of DNA	Sequences	Size (bp)
Na-Cl	5′-CCC AGC CAC GAG ATG ACT GA-3′	524
cotransporter	5′-TTG GAT TCC CAC TCC ATG CC-3′
Na-Dicarboxylate	5′-TGT TCC TTC TGC CCA TTT CCC-3′	634
cotransporter	5′-GTA GCA CAC GCA CAG GCT CAT-3′
Na-Phosphate	5′-TGG AGG AGG AGC AGA AGC CA-3′	533
cotransporter	5′-GGA TGT TGA AGG AGG CGA CC-3′
Na-Glucose	5′-TTCTTG GCG GGT CGG AGT ATG G-3′	553
cotransporter	5′-TGG CTG GGA ATG GCT CGC AT-3′
Na-K-2Cl	5′-TGC CCA CTA ATA CAA ATC GCT T-3′	686
cotransporter	5′-ACC TCC ACG AAC AAA CCC GT-3′
Urea Transporter	5′-TGA GAT AAA GGT GGA GAC CGC C-3′	690
	5′-ACA GCC ATA CAC TTG GCC GAT T-3′

Table 1. Alterations in renal function in control and postischemic kidneys

Parameters	Control	Postischemic
Urine flow (mL/day/kg body wt.)	90.19 ± 26.54	9.41 ± 5.91^*
GFR (l/day/kg body wt.)	1.53 ± 0.18	0.12 ± 0.12^*
Serum creatinine (mg/dL)	1.53 ± 0.11	4.04 ± 0.50^*
FENa (%)	1.77 ± 0.28	12.10 ± 6.55^*

Renal ischemia was induced by occlusion of bilateral renal arteries for 60 min. with a vascular clamp, and renal function was measured after 24 h. of reperfusion. Data are mean ± SE of 4 animals in each group.

^*p < 0.05 compared with the respective p control.

Figure 1. Alterations in fractional excretion of glucose (FE_Gl) and phosphate (FE_Pi). These changes were measured at 24–72 h of reperfusion following 60 min of ischemia in rabbits. Control samples were taken from sham-operated rabbits. Data are mean ± SE of 4 animals in each group.

Figure 2. Alterations in uptakes of D-glucose and phosphate in the presence of an inwardly-directed Na⁺ gradient by brush-border membrane vesicles isolated from control and postischemic kidney. Postischemic kidneys were subjected to 24–72 h of reperfusion following 60 min of ischemia. Membrane vesicles were suspended in a buffer containing 100 mM mannitol, 100 mM KCl, and 20 mM Hepes/Tris (pH 7.5), and were incubated in a buffer containing 100 mM mannitol, 100 mM NaCl, and 20 mM Hepes/Tris (pH 7.5). The concentration of substrate was 50 μM. Data are mean ± SE of 4 animals in each group.

Figure 3. RT-PCR analysis of expression of Na⁺-cotransporters in kidney cortex. Transcript levels of Na⁺-dicarboxylate, Na⁺-glucose and Na⁺-P_i were analyzed in cortex from control kidneys or kidneys subjected to 0 (ischemia), 24, 48, and 72 h of reperfusion following 60 min of ischemia. Data represent ratio to b-actin signals and are mean ± SE of 3 separate experiments. *p<0.05 compared with the control.

Figure 4. RT-PCR analysis of Na⁺-K⁺-2Cl, urea, and NaCl transporters in renal medulla. Transcript levels of these transporters were analyzed in medulla at control kidneys or kidneys subjected to 0 (ischemia), 24, 48, and 72 h of reperfusion following 60 min of ischemia. Data represent ratio to β-actin signals and are mean ± SE of 3 separate experiments.

Figure 5. Western analysis of AQP1 and AQP2. Kidneys were subjected to 0 (ischemia), 24, 48, and 72 h of reperfusion following 60 min of ischemia. Cortex and inner medulla of the kidneys were separated and membrane fractions were prepared as described in “Methods and Materials”. Polyclonal anti-rabbit antibodies for AQP1 and AQP2 were used for immunoblot analysis.