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Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney¹

CHUL WOO YANG^*, CAN LI^*,, JU YOUNG JUNG, SEOK JOON SHIN^*, BUM SOON CHOI^*, SUN WOO LIM^*, BO KYUNG SUN^*, YONG SOO KIM^*, JIN KIM^,2, YOON SIK CHANG^* and BYUNG KEE BANG^*

Cell Death Disease Research Center, Departments of
^* Internal Medicine, College of Medicine, and
Anatomy, The Catholic University of Korea, Seoul, Korea; and
Nephrology and Dialysis Unit, Affiliated Hospital, YanBian University Medical College, JiLin 133000, China

²Correspondence: Department of Anatomy, The Catholic University of Korea, 505 Banpo-Dong, Seocho-Ku, Seoul, 137-040, Korea. E-mail: jinkim{at}catholic.ac.kr

SPECIFIC AIMS

The aim of this study was to evaluate the protective effect of recombinant human erythropoietin (rHuEPO) against subsequent ischemia/reperfusion (I/R) injury in kidney and to evaluate the role of heat shock protein (HSP)70 in rHuEPO-induced renal protection.

PRINCIPAL FINDINGS

1. rHuEPO pretreatment modulates apoptotic signaling
In sham-operated rats, rHuEPO treatment significantly increased bcl-2 expression (2.7-fold) compared with rats without rHuEPO treatment. A similar effect was observed in rat kidneys with I/R injury. rHuEPO treatment significantly increased bcl-2 protein expression compared with rat kidneys with I/R injury alone (256±90% vs. 49±21%, P<0.05; Fig. 1 A). Caspase-3 proteolytic activity increased in the I/R group compared with the sham group (19.3±2.8 vs. 14.1±0.3 ng/µg protein, P<0.05), but pretreatment of rHuEPO decreased caspase-3 activity compared with those in the I/R group (13.3±0.4 vs. 19.3±2.8 ng/µg protein, P<0.05; Fig. 1B ).

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of pretreatment of rHuEPO on bcl-2 expression and caspase-3 activity in different groups. A) Immunoblot of bcl-2. Note the significant increase in bcl-2 protein in sham-operated rat kidney as well as ischemic rat kidneys with rHuEPO pretreatment compared with rats without rHuEPO treatment. #, P < 0.05, versus sham group; ##, P < 0.05, versus I/R group. S: sham-operated rats treated with saline; E+S: sham-operated rats treated with rHuEPO; I/R: rats subjected to ischemia followed by reperfusion; E+I/R: rats treated with rHuEPO before ischemia followed by reperfusion. B) Caspase-3 activity. In rat kidneys with I/R injury, caspase-3 activity increased significantly compared with sham or rHuEPO-pretreated sham rat kidneys. With rHuEPO pretreatment, caspase 3 activity decreased significantly. #, P < 0.05, versus sham group; ##, P < 0.05, versus I/R group.

2. rHuEPO pretreatment differentially regulates mitogen-activated protein kinase (MAPK)
In rat kidneys with I/R injury, extracellular-regulated kinase (ERK) or c-jun NH₂-terminal kinase (JNK) expression increased significantly compared with sham-operated rat kidneys (ERK, 3.2-fold; JNK, 6.7-fold, P<0.05). With pretreatment of rHuEPO, ERK expression was unchanged, but a significant reduction in JNK expression was observed compared with the I/R group (3.3-fold vs. 6.7-fold, P<0.05).

3. The renoprotective effect of rHuEPO is mediated by HSP70
rHuEPO treatment significantly increased HSP70 protein expression. Quercetin, a HSP70 inhibitor, decreased HSP70 expression induced by rHuEPO (Fig. 2 A; 120±9 vs. 512±26%, P<0.05) and subsequently, inhibited rHuEPO-induced renal protection (Fig. 2B-D ).

View larger version (72K): [in this window] [in a new window]   Figure 2. The effect of quercetin on rHuEPO-induced HSP70 expression and rHuEPO-induced renoprotection. A) The influence of quercetin (Q) on rHuEPO-induced HSP70 expression. Note the significant decrease in HSP70 protein expression after pretreatment of quercetin. #, P < 0.05, versus sham group; ##, P < 0.05, versus E+S group. S, Sham-operated rats treated with saline; Q+S, sham-operated rats treated with quercetin; E+S, rats treated with rHuEPO; Q+E+S, sham-operated rats treated with quercetin and rHuEPO. B) The impact of quercetin on renal function. Note that there is a significant increase in serum creatinine levels in the Q+E+I/R group compared with the E+I/R group. #, P < 0.05, versus sham group; ##, P < 0.05, versus I/R group; *, P < 0.05, versus E+I/R group. C, D) The influence of quercetin on tubular necrosis. Note that there is a significant increase in the necrosis score in the Q+E+I/R group compared with the E+I/R group. #, P < 0.05, versus sham group; ##, P < 0.05, versus I/R group; *, P < 0.05, versus E+I/R group. Q+I/R, Rats pretreated with quercetin before I/R injury; Q+E+I/R, rats treated with quercetin and rHuEPO before I/R injury.

CONCLUSIONS AND SIGNIFICANCE

The present study provides strong evidence that rHuEPO has a preventive effect against I/R injury in rat kidneys. In addition to previous studies that showed the role of rHuEPO in correcting anemia and improving renal tubular regeneration in postischemic kidney, our study expands knowledge about the role of rHuEPO as an inducer of ischemic tolerance in the kidney.

rHuEPO has an antiapoptotic effect against I/R injury. Our study demonstrated that rHuEPO pretreatment decreased caspase-3 activities and increased bcl-2 protein expression in rat kidneys with I/R injury. It is interesting that rHuEPO treatment of sham-operated rats also increased bcl-2 protein expression when compared with control kidneys. This finding suggests that rHuEPO treatment exerts its antiapoptotic effect by inducing bcl-2 protein expression in kidneys.

Our study demonstrates that pretreatment of rHuEPO differentially regulates MAPK expression. In rat kidneys with I/R injury, cell survival depends on ERK activation, and inhibition of JNK during ischemia ameliorates renal failure. In this study, rHuEPO pretreatment significantly decreased the expression of JNK without inhibiting activation of ERK expression in ischemic rat kidneys. These data suggest that pretreatment with rHuEPO controls thedynamic balance between ERK and JNK, and this regulation of MAPK favors protection against I/R injury.

The result of our study clearly demonstrates the role of HSP70 in rHuEPO-induced renoprotection. Quercetin treatment reduced EPO-induced HSP70 expression and subsequently eliminated the rHuEPO-induced renoprotective effect. This finding suggests that induction of HSP70 by rHuEPO is causally related to the observed protection in ischemic kidney.

Based on the above findings, we propose that HSP70 exerts its renoprotective effects by modulating apoptotic cell signaling and stress kinases (Fig. 3 ). The ability of rHuEPO to induce HSP70 provides a new avenue for therapy to prevent renal damage in I/R injury.

View larger version (18K): [in this window] [in a new window]   Figure 3. Diagram showing role of HSP70 in EPO-induced renoprotection. The rHuEPO treatment increases HSP70 protein, and overexpressed HSP70 exerts its renoprotective effect via modulating apoptotic signaling and MAPK activation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1191fje; doi: 10.1096/fj.02-1191fje

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