Evolution of renal function and Na+, K +-ATPase expression during ischaemia-reperfusion injury in rat kidney.

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na+, K+-ATPase activity, alpha1 and beta1 subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na+, K+-ATPase alpha1 subunit abundance increased in IR24 and IR48 cortical tissue and beta1 subunit decreased in IR48. In IR24 medullary tissue, alpha1 abundance increased and returned to control values in IR48 while beta1 abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in alpha1 mRNA in cortex and medulla that normalised after 24 h of reperfusion. beta1 mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult alpha1 and beta1 protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na+, K+-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.

Effect of acetaminophen on the membrane anchoring of Na+, K+ATPase of rat renal cortical cells.

In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na(+), K(+)ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na(+), K(+)ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na(+), K(+)ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both alpha(1) and beta(1) subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na(+), K(+)ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na(+), K(+)ATPase.

Fibronectin expression in proximal tubules from ischemic rat kidneys without reperfusion.

The expression of fibronectin (FN), one of the extracellular matrix proteins, was studied in isolated renal proximal tubules in a in vivo rat model of unilateral renal ischemia without reperfusion. FN is involved in cell-extracellular matrix interactions and defective cell-extracellular matrix interactions have been hypothesized to contribute to ischemic renal failure. The expression of FN was investigated by reverse transcription-polymerase chain reaction (RT-PCR), Elisa and Western blot. Isolated proximal tubules from control and post-ischemic rat kidneys were used. ATP, intracellular calcium content, and alkaline phosphatase were also measured to describe the effects associated to 40 min of ischemia. Control tubules expressed FN. Forty minutes of ischemia promoted diminished ATP levels and phosphatase alkaline activity, and increased intracellular calcium in isolated proximal tubules. An increased abundance of FN was observed by ischemic tubules as compared with control tubules. To determine quantitatively the value of FN content, ELISA method was performed. The ischemic tubules also expressed higher amount of FN mRNA. Three amplification products were obtained from both ischemic and control proximal tubular cDNA. The relative amounts of each of the obtained products were the same, strongly suggesting that the augmentation of the FN gene transcription during ischemia is not associated to a modification in the splicing pattern. Moreover, this expression is increased after 40 min of ischemia, not followed by reperfusion.

Reversal of benzodiazepine-induced renal vasculature relaxation with flumazenil.

The effects of the central-type benzodiazepine receptor antagonist flumazenil on renal vascular tone and its ability to reverse the benzodiazepine-induced vasodilation were investigated. The isolated and perfused rat kidney model was used. Flumazenil was unable to modify renal vascular resistance under basal conditions and in noradrenaline-pretreated kidneys. Relaxation induced by diazepam or clonazepam of noradrenaline-preconstricted renal vasculature was blunted by 10 microM flumazenil. These results suggest that central-type benzodiazepine receptors could be involved in benzodiazepine-induced renal vasodilation.

Renal function and cortical (Na(+)+K(+))-ATPase activity, abundance and distribution after ischaemia-reperfusion in rats.

The effects of ischaemic injury and reperfusion on renal function, cortical ATP content, alkaline phosphatase activity and (Na(+)+K(+))-ATPase activity and abundance in cortical homogenates and isolated basolateral and apical membranes were examined. Rats were submitted to 5 or 40 min of right renal artery occlusion and 60 min of reperfusion. Renal function of the ischaemic-reperfused kidney was studied by conventional clearance techniques. Our results show that 1 h of reperfusion after a short period of renal ischaemia (5 min) allows the complete restoration of the biochemical features of cortical cells and functional properties of the injured kidney. A longer period of ischaemia, such as 40 min, followed by 1 h of reperfusion showed functional and biochemical alterations. ATP recovered from 26% after 40 min of ischaemia to 50% of control values after 1 h reperfusion. However, renal function was strongly impaired. Brush border integrity was compromised, as suggested by AP excretion and actin appearance in urine. Although total cortical (Na(+)+K(+))-ATPase activity was not different from controls, its distribution in isolated apical and basolateral membranes was abnormal. Remarkably, we detected an increase in alpha-subunit protein abundance that may suggest that (Na(+)+K(+))-ATPase synthesis is promoted by ischaemia-reperfusion. This increase may play an important role in the pathophysiology of ischaemic acute renal failure.