Prevention of Kidney Ischemia/Reperfusion-induced Functional Injury and JNK, p38, and MAPK Kinase Activation by Remote Ischemic Pretreatment

Abstract

MAPK activities, including JNK, p38, and ERK, are markedly enhanced after ischemia in vivoand chemical anoxia in vitro. The relative extent of JNK, p38, or ERK activation has been proposed to determine cell fate after injury. A mouse model was established in which prior exposure to ischemia protected against a second ischemic insult imposed 8 or 15 days later. In contrast to what was observed after 30 min of bilateral ischemia, when a second period of ischemia of 30- or 35-min duration was imposed 8 days later, there was no subsequent increase in plasma creatinine, decrease in glomerular filtration rate, or increase in fractional excretion of sodium. A shorter period of prior ischemia (15 min) was partially protective against subsequent ischemic injury 8 days later. Unilateral ischemia was also protective against a subsequent ischemic insult to the same kidney, revealing that systemic uremia is not necessary for protection. The ischemia-related activation of JNK and p38 and outer medullary vascular congestion were markedly mitigated by prior exposure to ischemia, whereas preconditioning had no effect on post-ischemic activation of ERK1/2. The phosphorylation of MKK7, MKK4, and MKK3/6, upstream activators of JNK and p38, was markedly reduced by ischemic preconditioning, whereas the post-ischemic phosphorylation of MEK1/2, the upstream activator of ERK1/2, was unaffected by preconditioning. Pre- and post-ischemic HSP-25 levels were much higher in the preconditioned kidney. In summary, post-ischemic JNK and p38 (but not ERK1/2) activation was markedly reduced in a model of kidney ischemic preconditioning that was established in the mouse. The reduction in JNK and p38 activation can be accounted for by reduced activation of upstream MAPK kinases. The post-ischemic activation patterns of MAPKs may explain the remarkable protection against ischemic injury observed in this model. MAPK activities, including JNK, p38, and ERK, are markedly enhanced after ischemia in vivoand chemical anoxia in vitro. The relative extent of JNK, p38, or ERK activation has been proposed to determine cell fate after injury. A mouse model was established in which prior exposure to ischemia protected against a second ischemic insult imposed 8 or 15 days later. In contrast to what was observed after 30 min of bilateral ischemia, when a second period of ischemia of 30- or 35-min duration was imposed 8 days later, there was no subsequent increase in plasma creatinine, decrease in glomerular filtration rate, or increase in fractional excretion of sodium. A shorter period of prior ischemia (15 min) was partially protective against subsequent ischemic injury 8 days later. Unilateral ischemia was also protective against a subsequent ischemic insult to the same kidney, revealing that systemic uremia is not necessary for protection. The ischemia-related activation of JNK and p38 and outer medullary vascular congestion were markedly mitigated by prior exposure to ischemia, whereas preconditioning had no effect on post-ischemic activation of ERK1/2. The phosphorylation of MKK7, MKK4, and MKK3/6, upstream activators of JNK and p38, was markedly reduced by ischemic preconditioning, whereas the post-ischemic phosphorylation of MEK1/2, the upstream activator of ERK1/2, was unaffected by preconditioning. Pre- and post-ischemic HSP-25 levels were much higher in the preconditioned kidney. In summary, post-ischemic JNK and p38 (but not ERK1/2) activation was markedly reduced in a model of kidney ischemic preconditioning that was established in the mouse. The reduction in JNK and p38 activation can be accounted for by reduced activation of upstream MAPK kinases. The post-ischemic activation patterns of MAPKs may explain the remarkable protection against ischemic injury observed in this model. Ischemic injury to brain, heart, and kidney is associated with high morbidity and mortality. Improving the ability of these organs to tolerate ischemic injury would have important implications. Ischemic insults are often recurrent in patients. In the setting of loss of renal blood flow autoregulation that characterizes the post-ischemic kidney (1Conger J. Adv. Renal Replace. Ther. 1997; 4 Suppl. 1: 25-37Google Scholar), it might be expected that the post-ischemic patient would be more susceptible to a second insult. It has been reported in animals, however, that prior acute renal failure induced by toxins can confer resistance to subsequent insults, although this is not a universal finding (2Honda N. Hishida A. Ikuma K. Yonemura K. Kidney Int. 1987; 31: 1233-1238Abstract Full Text PDF PubMed Scopus (84) Google Scholar). Zager et al. (3Zager R.A. Baltes L.A. Sharma H.M. Jurkowitz M.S. Kidney Int. 1984; 26: 689-700Abstract Full Text PDF PubMed Scopus (105) Google Scholar) have focused on early time points after an initial ischemic exposure and have concluded that the rat is not more susceptible to a second ischemic episode when timed at or near the peak of functional deficit after the first. The authors further concluded that “a modicum of protection appears to exist, possibly due to renal failure-induced increments in solute load.” Proximal tubules isolated 24 h (but not 15 min or 4 h) after an ischemic episode are protected against hypoxia, reactive oxygen species, or calcium ionophore in vitro (4Zager R.A. Burkhart K.M. Gmur D.J. Lab. Invest. 1995; 72: 592-600PubMed Google Scholar, 5Zager R.A. Iwata M. Burkhart K.M. Schimpf B.A. Kidney Int. 1994; 45: 1760-1768Abstract Full Text PDF PubMed Scopus (75) Google Scholar). The lack of protection at 4 h and the induction of protection by inducing azotemia in vivo or adding urine to the tubulesin vitro led the authors to conclude that the protection is due to the uremic environment. Subsequently protection was found in tubules obtained from obstructed kidneys even when the tubules had not been exposed to a uremic environment (6Zager R.A. Kidney Int. 1995; 47: 628-637Abstract Full Text PDF PubMed Scopus (31) Google Scholar). In a recent study (7Lee H.T. Emala C.W. Am. J. Physiol. Renal Physiol. 2000; 278: F380-F387Crossref PubMed Google Scholar), four cycles of 8 min (but not 4 or 6 min) of ischemia, separated by 5 min of reperfusion, conferred protection against a subsequent 45-min ischemic exposure immediately after the four cycles. In another recent study, four cycles of 4 min of ischemia, separated by 11 min of reperfusion, followed 5 min later by 45 min of ischemia, resulted in fewer dead renal cells, but no difference in creatinine at 6 h post-ischemia with this preconditioning protocol (8Jefayri M.K. Grace P.A. Mathie R.T. Br. J. Urol. Int. 2000; 85: 1007-1013Crossref PubMed Google Scholar). It had previously been shown that there were no morphological differences with this preconditioning protocol if the time delay between the cycles and the prolonged ischemia was 30 min (9Islam C.F. Mathie R.T. Dinneen M.D. Kiely E.A. Peters A.M. Grace P.A. Br. J. Urol. 1997; 79: 842-847Crossref PubMed Google Scholar). Thus, controversy remains as to the conditions under which preconditioning occurs in the kidney in response to prior ischemia. In addition, the reported experiments are limited to the study of short intervals of time between the preconditioning protocol and the tested period of subsequent ischemia. One approach to the design of therapies to protect the kidney against ischemic injury is to establish a model in which endogenous mechanisms result in protection and then identify those processes. A model in mice would be particularly useful given the plethora of transgenic models that exist, which would facilitate exploration of the role of a particular protein in the protection. Furthermore, we wanted to establish a model in which the acute cellular and molecular response to the initial event had subsided, reducing the “background” genetic response so that it would be more possible to identify an endogenous protective factor or factors. Our experiments demonstrate that 30 min of bilateral renal ischemia, resulting in significant increases in blood urea nitrogen and creatinine, leads to protection of the mouse kidney against a subsequent ischemic insult 8 or 15 days later, even when the second ischemic period is extended to 35 min. Graded levels of time of initial ischemia resulted in graded levels of protection 8 days later. This protection is unrelated to systemic effects of transient uremia since unilateral ischemia is also associated with protection under conditions in which there is very little increase in systemic blood urea nitrogen or creatinine. Preconditioning results in prevention of medullary congestion. With varying times of initial ischemia, the protection afforded 8 days later correlates with the level of sustained elevations in HSP-251 (HSP-27 in rat) protein levels prior to the second ischemic period, but not superoxide dismutase or HSP-72 levels. The MAPKs have been implicated in post-ischemia/reperfusion cell survival, necrosis, and apoptosis (10Pombo C.M. Bonventre J.V. Avruch J. Woodgett J.R. Kyriakis J.M. Force T. J. Biol. Chem. 1994; 269: 26545-26551Abstract Full Text PDF Google Scholar, 11Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5032) Google Scholar, 12Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar). MAPKs mediate the response of cells to a wide variety of physiological and stress-related stimuli, including ultraviolet light, heat shock, ischemia, oxygen free radicals, and hyperosmolality. It has been proposed that activation of JNK and p38 kinases contributes to cell death, whereas activation of ERK1/2 contributes to protection against cell injury in multiple organs (11Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5032) Google Scholar, 13Safirstein R. DiMari J. Megyesi J. Price P. Semin. Nephrol. 1998; 18: 519-522PubMed Google Scholar). We examined whether the effect of ischemic preconditioning to protect the kidney is associated with corresponding changes in stress kinases and ERKs as well as their upstream activating kinases, which might account for the functional protection observed. Post-ischemic activation of JNK and p38 is markedly mitigated in the preconditioned kidneys, as is the post-ischemic activation of the MAPK kinases MKK7, MKK4, and MKK3/6, upstream activators of JNK and p38. By contrast, the post-ischemic activation of MKK3/4, the MAPK kinases responsible for activating ERK1/2, is unaltered by preconditioning. All experiments were performed with male BALB/c mice (Charles River Laboratories) weighing 20–25 g. Mice were allowed free access to water and standard mice chow. Blood was drawn, and base-line levels of serum creatinine and blood urea nitrogen were determined 2 days before surgery. Animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally) and administered 1 ml of 0.9% NaCl (37 °C) on the of was at Animals were were exposed Mice in and were to bilateral renal ischemia by renal with Animals in and surgery. The were ischemia or surgery. 5 15 or 30 and the were and of the kidneys was Animals were exposed to 30 or 35 and min of bilateral ischemia on 8 or 30 min of ischemia on 15 and and or bilateral ischemia ischemia, 5 ischemia ischemia, 15 ischemia ischemia bilateral ischemia, 35 min ischemia, 35 min ischemia bilateral ischemia kidney kidney on kidney ischemia kidney kidney on kidney ischemia kidney kidney on kidney ischemia kidney kidney on kidney ischemia kidney kidney ischemia on ischemia as times of ischemia were 30 min. in of mice in in a as times of ischemia were 30 min. in of mice in Mice in unilateral ischemia for 30 min. In the kidney was on whereas in the kidney was on at the time of the second ischemia. In the kidney was ischemic on the kidney was on and the kidney was ischemic on In the kidney was ischemic for 30 min on that kidney was and the kidney was ischemic on In ischemia to kidney on was followed by ischemia to the kidney on and bilateral ischemia was imposed on of were on or 8 after the or 24 or h after the second ischemic were in nitrogen and for or were in and in for of blood were from the at the times in the and urine creatinine, and blood urea nitrogen were a a or a urea determine glomerular filtration and fractional excretion of urine was for 24 were from kidneys as previously (10Pombo C.M. Bonventre J.V. Avruch J. Woodgett J.R. Kyriakis J.M. Force T. J. Biol. Chem. 1994; 269: 26545-26551Abstract Full Text PDF Google Scholar). were separated on a or and then to an were with against MEK1/2, and ERK1/2, and p38 by A. HSP-72 and superoxide dismutase with were by the from mouse kidney were for protein prior to with a against JNK as a protein in were with protein and were performed as (10Pombo C.M. Bonventre J.V. Avruch J. Woodgett J.R. Kyriakis J.M. Force T. J. Biol. Chem. 1994; 269: 26545-26551Abstract Full Text PDF Google Scholar) with as min at 30 the were with were by with and to The corresponding to were of the was by were the with at and then with M. Bonventre J.V. Am. J. Physiol. 1999; Google Scholar) for min. In the was in to and with and All results are as A was as of as in Animals were exposed to or or 30 min of bilateral renal ischemia levels of creatinine base-line levels 24 h post-ischemia when the ischemic period was 30 min. By 5 after ischemia, creatinine levels were from those at in 8 after the initial ischemia or a subsequent 30 min of bilateral ischemia was The increase in creatinine 24 h later was with the of time of the prior ischemia. was no significant increase in serum creatinine at 24 and h after the second ischemic period in that were previously exposed to 30 min of ischemia. The patterns of in blood urea nitrogen after the and second ischemic those in creatinine in these and subsequent experiments The glomerular filtration was markedly the after 30 min of ischemia, but to days of 1 fractional excretion of sodium was markedly the of reperfusion, but to levels by days of 1 A subsequent second ischemic period resulted in no decrease in glomerular filtration or increase in fractional excretion of sodium. was found in kidneys from h after 30 min of bilateral ischemia. were found in the outer The kidneys of were the of tubules was tubules were and tubules were and kidneys were examined h after the second ischemia, the kidneys had no significant outer medullary whereas there was very significant congestion in the outer of examined after their exposure to ischemia much of cells, and cell in the outer of preconditioned with and the ischemic period was 30 min and the second was extended to 35 min serum creatinine not increase after the of the of time between bilateral ischemic was to 15 days serum creatinine by 24 h after the second ischemic insult however, the increase was that observed in that had been previously on of prior ischemia for 30 min on serum creatinine after 30 min of a second period of ischemia on were to or 30 min of ischemia days after the were to bilateral ischemia for 30 min. the of ischemia or surgery. and whether ischemia was protective of systemic effects of were exposed to or unilateral kidney ischemia and for 30 min on We that the of a kidney might mechanisms to protection against a subsequent ischemic exposure to a kidney ischemic A. J. Lab. 1984; Google Scholar). or 8 days later, the kidney was and at 8 the kidney was ischemic for 30 min unilateral ischemia protected that kidney against subsequent ischemia even in the of an increase in serum creatinine the kidney was in for 8 however, the protection was was when the kidney was at 2 days In the kidney was ischemic on the kidney was ischemic for 30 min on and kidneys were ischemic on was protection against ischemic injury under these with no increase in serum creatinine 24 h after the bilateral ischemia whether unilateral ischemia protection on the kidney, in the kidney was ischemic for 30 min on and on and the kidney was ischemic for 30 min on later, the serum creatinine was markedly that unilateral ischemia not confer protection on the kidney. We whether or superoxide dismutase protein levels with the of resistance to subsequent ischemia. The levels of HSP-25 at 8 days post-ischemia with time of ischemia well with the extent of protection against functional after the second ischemic period the second ischemic period, HSP-25 levels markedly to higher levels in the preconditioned kidneys whereas HSP-72 levels were no whether the was ischemia or The protein levels of superoxide dismutase were not markedly at 8 days post-ischemia not The JNK of MAPKs as is markedly as early as 5 min after at but remains at high levels to 2 h post-ischemia (10Pombo C.M. Bonventre J.V. Avruch J. Woodgett J.R. Kyriakis J.M. Force T. J. Biol. Chem. 1994; 269: 26545-26551Abstract Full Text PDF Google Scholar). In these we have found that JNK levels at h of In previously exposed to ischemia, subsequent ischemia on 8 or 15 resulted in much activation of was in post-ischemic kidneys not previously exposed to ischemia 8 activation was determined by with the a was with reduction of the increase in at and h post-ischemia in the exposed to ischemia 8 days previously 8 post-ischemic p38 activation was markedly reduced in kidneys previously exposed to ischemia ERK1/2 activation 8 days after the initial ischemic but in contrast to JNK and p38, there was no effect of prior ischemia on post-ischemic ERK phosphorylation on 8 whether activation of JNK, p38, and ERK1/2 is by changes in activation patterns of upstream of these kinases, we the response to ischemia of JNK and possibly and ERK1/2) in kidneys previously exposed to ischemia or The post-ischemic increase in phosphorylation of MKK7, MKK4, and was much in the preconditioned kidney in the kidney. By contrast, post-ischemic phosphorylation of was not by preconditioning. phosphorylation of at h after ischemia, phosphorylation of MKK4, MKK3/6, and at Ischemic preconditioning may to important an endogenous mechanisms to protect against injury. the and for of that might be implicated in this it is necessary to establish a mouse model in which that protection is very and We to establish a model that the of to the initial at the time when we study the mechanisms of protection. This the associated with the functional of of of that occurs days of ischemia. we have found fewer in at 8 days post-ischemia not mechanisms of protection afforded by to injury may to for these to ischemic injury. ischemic preconditioning in the kidney has been Zager et al. (3Zager R.A. Baltes L.A. Sharma H.M. Jurkowitz M.S. Kidney Int. 1984; 26: 689-700Abstract Full Text PDF PubMed Scopus (105) Google Scholar) reported that min of bilateral ischemia in the rat not confer enhanced to a second ischemic insult imposed at the peak of functional deficit after the ischemia. In a subsequent R.A. Jurkowitz M.S. Am. J. Physiol. Google Scholar), this reported that min of renal ischemia decrease renal resistance to a subsequent ischemic Renal cells isolated from 24 h after 35 min of ischemia were protected against injury as by R.A. Iwata M. Burkhart K.M. Schimpf B.A. Kidney Int. 1994; 45: 1760-1768Abstract Full Text PDF PubMed Scopus (75) Google Scholar). Proximal tubules were not protected if were isolated from 15 min of bilateral ischemia when uremia was not Furthermore, the of a kidney markedly after unilateral ischemia, a in which uremia not et al. (9Islam C.F. Mathie R.T. Dinneen M.D. Kiely E.A. Peters A.M. Grace P.A. Br. J. Urol. 1997; 79: 842-847Crossref PubMed Google Scholar) found that cycles of renal followed by not functional or morphological protection of rat kidney from a subsequent ischemic insult 30 min later. et J. A. Renal 1999; 21: PubMed Scopus Google Scholar) observed a serum creatinine level after 24 h of in with ischemia for 2 min or of ischemia by 5 min but not of ischemia of Our experiments that mice that had from 30 min of bilateral renal ischemia were protected against subsequent ischemia imposed 8 or 15 days after the ischemic The protection against functional injury 8 days after ischemia was a very of fractional excretion of sodium was unaffected by 30 min of ischemia. By contrast, it to in post-ischemic kidneys not previously exposed to ischemia. The preconditioning protocol is the to protect the mouse kidney against ischemic injury. is not on the of a uremic systemic environment since 15 min of initial ischemia resulted in protection against subsequent ischemia an increase in blood urea A effect the kidney is responsible for protection against subsequent ischemia since protection was observed in the same kidney that sustained unilateral ischemia, but not in the kidney. Our finding that the protection was when the kidney was on 2 with 8 is with prior of the effects of the of the kidney on the functional of a ischemic kidney A. J. Lab. 1984; Google Scholar). In the heart, although preconditioning is a very to post-ischemic the protective effects of preconditioning are transient and for a short period of A of has been observed in R.A. 1997; PubMed Scopus Google Scholar), 24 h after the preconditioning in and In study, the protection in the mouse kidney after ischemia was to 15 days after the initial ischemic period, when but not of the and stress associated with ischemia have been The of protection against the second ischemic insult after a ischemic period of varying time duration with levels of heat protein at 8 days may decrease of A. N. 1999; PubMed Scopus Google Scholar), reducing and congestion in the outer resulting in injury to the outer medullary It has been that HSP-25 against or heat stress by the J. J. Google Scholar). The is markedly in the after ischemia B.A. R. A. Am. J. Physiol. Google Scholar, R. Bonventre J.V. Am. J. Physiol. 1997; Google Scholar). important role of HSP-25 is that of a molecular in a after of physiological conditions post-ischemia M. M. J. J. 1997; PubMed Scopus Google Scholar). HSP-25 can also increase cellular levels of P. J. PubMed Scopus Google Scholar), which can protect the cell against stress activation R. DiMari J. Megyesi J. Price P. Semin. Nephrol. 1998; 18: 519-522PubMed Google Scholar), and apoptosis P. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HSP-25 levels increase in the and outer medullary tubules of rat kidneys post-ischemia R. R. J. Am. Nephrol. 2000; PubMed Google Scholar). By contrast to changes in dismutase protein implicated in the of preconditioning in the N. N. T. M. J. 1998; Full Text PDF PubMed Scopus Google Scholar), not with protection of the kidney in Our of increases in JNK, p38, and ERK1/2 activation with ischemia is with results from (10Pombo C.M. Bonventre J.V. Avruch J. Woodgett J.R. Kyriakis J.M. Force T. J. Biol. Chem. 1994; 269: 26545-26551Abstract Full Text PDF Google Scholar) and M. J. A. R. A. P. 79: PubMed Scopus Google Scholar) in the kidney and In the heart, have that activation of p38 is important for preconditioning A. J.M. 2000; PubMed Scopus Google Scholar), whereas have that activation is and that a reduced level of activation is important for preconditioning R.J. M.S. J. 2000; PubMed Scopus Google Scholar). It was previously not the upstream kinases responsible for activation of the MAPKs are by kidney ischemia. Our that results in the activation of and are activators of p38, although the physiological of as an activator of p38 is This activation is reduced in kidneys previously exposed to ischemia in a that that of p38 JNK is by phosphorylation on and by and R.J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). results in and This post-ischemic activation is reduced in kidneys previously exposed to ischemia in a that that of p38 and JNK activation at h after ischemia and JNK This that post-ischemic JNK activation may be more In contrast to the upstream activators of the stress kinases, post-ischemic activation is not reduced in the preconditioned kidney. The increase in with a increase in ERK1/2 activation post-ischemia/reperfusion in the setting of reduced stress activation may explain the protection with preconditioning in the kidney. It has been proposed in (11Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5032) Google Scholar) and cells Davis R. Am. J. Physiol. 1999; Google Scholar) that the relative extent of JNK, p38, and ERK activation may determine cell with JNK activation associated with cell and ERK JNK has been implicated in the P. J. A. R.A. Davis R.J. Science. 2000; PubMed Scopus Google Scholar), and activation of the JNK has been reported to be important for in response to ischemia A. R.A. 1999; PubMed Scopus Google Scholar). The functional protection we observed is associated with a reduction in outer medullary congestion which may be to associated with the decrease in activation of and p38. JNK and p38 activation the of J. Davis R.J. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and R. P. K. M. Biol. 1999; PubMed Scopus Google Scholar, Biol. 1997; PubMed Google Scholar, R. J. Invest. 1999; PubMed Scopus Google Scholar), in can in the of the outer with associated activation and to injury. The reduction in post-ischemic outer medullary congestion in the kidney previously exposed to ischemia for an important effect of preconditioning to and In summary, we have that the mouse kidney is protected against injury to 15 days after an initial ischemic insult. This points to endogenous mechanisms that can be by the kidney to protect against ischemic injury. This protection is associated with prevention of outer medullary vascular congestion and of the increase in JNK and p38 with no effect on ERK1/2 The effects of ischemic on MAPK activation can explain the changes observed in MAPK activation protection is after of the initial of the have subsided, it is that a or approach to of that are or that are be This then to therapies that be in ischemic acute renal heat protein protein MAPK protein protein