promising effects of ischemic preconditioning in renal transplantation

Kidney International, Vol. 61 (2002), pp. 2218–2227

DIALYSIS – TRANSPLANTATION

Promising effects of ischemic preconditioningin renal transplantation

JOAN TORRAS, IMMACULADA HERRERO-FRESNEDA, NURIA LLOBERAS, MARTA RIERA,JOSEP Ma CRUZADO, and JOSEP Ma GRINYO

Department of Nephrology and Laboratory of Experimental Nephrology, Hospital of Bellvitge, L’Hospitalet,Department of Medicine, University of Barcelona, Ciutat Sanitaria i Universitaria de Bellvitge, Barcelona, Spain

NO. As renal DNA oxidative injury paralleled NO, increasingPromising effects of ischemic preconditioning in renal trans-plantation. with prolongation of reperfusion, it may account for the disap-

Background. Ischemic preconditioning, a phenomenon in- pearance of preconditioning. Finally, the one-cycle precondi-duced by brief ischemia and reperfusion periods, renders an tioning schedule offered an effective functional and histologicalorgan tolerant to subsequent prolonged ischemia. This study protection against cold preservation damage in rat renal trans-evaluated different schedules of preconditioning the kidney to plantation.assess the role of nitric oxide (NO) and determine the effects Conclusions. Fifteen minutes of warm ischemia and 10 min-of preconditioning on kidney transplantation. utes of reperfusion in the kidney is the most suitable one-cycle

Methods. In study design A, to determine the optimum pro- schedule for preconditioning since it protects from both warmcedure of preconditioning, one-cycle schedules were assayed by and cold ischemia. The beneficial effect of preconditioning isoccluding/releasing renal pedicles according to various warm related to the local production of NO, and we believe it hasischemic (5, 10, 15, 20 min) and reperfusion (10, 20, 40 min) promising therapeutic value in clinical renal transplantation.windows in Sprague-Dawley rats. Thereafter, warm renal isch-emia was induced by clamping both pedicles for 40 minutes.Design B used the most suitable schedule found from the first

In renal transplantation, the deleterious effect of de-study to obtain several groups, using either a direct nitric oxidelayed graft function secondary to ischemia-reperfusiondonor (spermine NONOate) or two nitric oxide synthase

(NOS) blockers (L-NAME and aminoguanidine), to determine damage may act to trigger or amplify host allo-responsewhether NO mediates in renal preconditioning. To establish and influence both the acute rejection occurrence [1]whether preconditioning reduces cold preservation damage, in and late chronic changes [2]. Thus, protective maneuversDesign C the optimum preconditioning schedule was used in at the time of transplantation should clearly benefit thesyngeneic Lewis rats where preconditioned and non-precondi-

fate of the organ.tioned kidneys were transplanted after five hours of cold stor-Ischemic preconditioning, a phenomenon induced byage in Euro-Collins solution.

Results. The best preconditioning schedule consisted of 15 brief ischemia and reperfusion periods, renders an organminutes of warm ischemia and 10 minutes of reperfusion (Prec more tolerant to subsequent sustained ischemia-reper-15/10), since it was the only schedule that offered both func- fusion [3]. This phenomenon has been mainly studiedtional and histological protection. The NO donor reproduced and characterized in the heart [3, 4], but it has also beenthe ischemic preconditioning. Non-selective NOS blockade

described in the liver [4–6], the small intestine [7, 8] andabolished the preconditioning and exacerbated ischemic dam-the brain [9]; however, little information available in theage, which was overcome by the addition of the NO donor.kidney. A report in the early 1980s focused on the lateSelective blocking of inducible NOS also abolished the effects

of preconditioning. Renal NO increased at the end of precondi- acquisition of resistance against ischemic injury throughtioning in the Prec 15/10 group. Prolongation of the reperfusion the induction of intrinsic antioxidant enzymes by a previ-window (20 or 40 min) abolished the preconditioning protec- ous episode of short ischemia [10]. This is a protein-tion, although it was associated with a further increase in renal

dependent mechanism similar to the late phase of pre-conditioning described in the heart [11]. More recently,some studies concerning early protection of renal tissueKey words: ischemia-reperfusion, warm and cold renal ischemia, nitric

oxide, organ preservation, delayed graft function, kidney preservation. by ischemic preconditioning have been drafted [12–14]with contradictory results. All these studies used a four-

Received for publication July 20, 2001cycle preconditioning schedule similar to that classicallyand in revised form January 7, 2001

Accepted for publication January 8, 2002 applied in the heart [11, 15]. Using an easier one-cycleschedule, preconditioning has been achieved in some 2002 by the International Society of Nephrology

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Torras et al: Ischemic preconditioning in the kidney 2219

organs [6, 8] and our preliminary experiment ensured the kidney was immediately washed with Euro-Collinssolution (EC; 4 mL, 4�C, 20 mL/h flow rate). Thereafter,protection of the kidney against warm ischemia [16].

Although the mechanisms of ischemic preconditioning the kidney was either transplanted immediately or pre-served in EC at 4�C before transplantation. For het-are unclear, potential mediators include heat shock pro-

teins [17, 18], endothelin [19], nitric oxide (NO) [15, 20] erotopic renal transplantation, the host left kidney wasnephrectomized, the donor artery and vein were anasto-and adenosine [5, 6, 9, 14]. The production of NO affects

organ integrity in response to various challenges. In this mosed end-to-side to the receptor aorta and cava, andthe ureter was anastomosed end-to-end. Re-anastomosissense, l-arginine and NO donors provide significant pro-

tection in ischemia-reperfusion in several organs [21, 22], took no more than 30 minutes and total surgical time didnot exceed 60 minutes. The grafts that did not reperfusewhile exogenous NO administration may also mimic

ischemic preconditioning [23]. In addition, inhibition of correctly were rejected.After surgery, animals were housed in a room kept atNO can induce or aggravate most of the alterations elic-

ited by ischemic damage [24, 25]. In the kidney, NO is constant temperature with a 12 h/12 h light/dark cycle.Rats were fed a standard diet and tap water ad libitum.involved in ischemia-reperfusion injury [22, 26], but few

data are available regarding its role in ischemic precondi- Before surgery and on days 1, 2, 3 and 7, animals wereweighed and a blood sample was obtained from the tailtioning.

Thus, we first aimed to assess whether preconditioning vein for serum creatinine (SCr) measurement (�mol/L),which was performed by an autoanalyzer (Beckman In-can be induced in the kidney and if so, which is the

optimum schedule of preconditioning. To discover the struments, Palo Alto, CA, USA) following Jaffe’s reac-tion. Rats were killed under ketamine anesthesia, bloodmechanisms of preconditioning, we also studied the role

of NO in kidney preconditioning by treating with NO was obtained by aortic puncture and the kidneys wereprocessed for tissue studies.donors and by inhibiting nitric oxide synthase (NOS).

Finally, since the major interest of renal ischemic precon-Histological studiesditioning should be its applicability to clinical trans-

plantation, we determined whether this protective effect For conventional histology, coronal 1- to 2-mm thickslices of the kidney were fixed in 4% formaldehyde andcould be extended to kidney preservation.embedded in paraffin, and 3- to 4-�m thick tissue sectionswere stained with hematoxylin and eosin (H&E), and

METHODSby the periodic acid-Schiff (PAS) methods. Light micros-

Animals and surgical procedure copy sections were examined by a pathologist who wasblinded to the treatment groups. Samples were analyzedAll procedures followed the Guidelines of the Euro-

pean Community Committee on Care and Use of Labo- for tubular cell necrosis, tubular dilation, intratubularcell detachment, interstitial edema and interstitial cellu-ratory Animals and Good Laboratory Practice. Warm

ischemia studies were carried out in male Sprague-Daw- lar infiltrate. Abnormalities were graded using a semi-quantitative scale from 0 to 4�, in which 0 denotes noley rats (250 to 300 g body wt) from our in-house breed-

ing facility. Under intramuscular (IM) anesthesia with a abnormalities, 1� � changes affecting �25% of the sam-ple, 2� � changes affecting 25 to 50% of the sample,mixture of ketamine (75 mg/kg), atropine (0.05 mg/kg)

and diazepam (5 mg/kg), a midline laparotomy was per- 3� � changes affecting 50 to 75% of the sample and4� � changes affecting �75% of the sample.formed, renal vessels were dissected, and warm ischemic

damage was induced by cross-clamping both renal pedi-Study designcles for 40 minutes. During ischemia, animals were kept

at 37�C. Preliminary data from our laboratory indicated Design A. To study the effect of ischemic precondi-tioning on warm ischemia and obtain the most suitablethat cross-clamping of both the renal artery and vein for

40 minutes produced consistent ischemic injury. preconditioning schedule, two sets of experiments wereperformed. On one hand, four warm ischemic precondi-Cold ischemia studies were carried out following a

renal transplantation (Tx) model, in which donors and tioning times (5, 10, 15 and 20 min) with a constantreperfusion time of 10 minutes prior to the 40 minutesrecipients were inbred male Lewis rats (250 g body

weight; Charles River, Sta. Perpetua de la Mogoda, Bar- of warm ischemia were analyzed. For this purpose, fortyanimals were classified in five groups: (1) the ISC groupcelona, Spain) and surgery was performed as described

elsewhere [27, 28]. For kidney harvesting and trans- had no preconditioning and 40 minutes of warm ischemia(N � 8); (2) the Prec 5/10 group had 5 minutes of warmplantation, anesthesia was induced and maintained by

an IM injection of Droperidol (neuroleptoanalgesic; 0.5 ischemia, 10 minutes of reperfusion, and 40 minutes ofwarm ischemia (N � 8); (3) the Prec 10/10 group hadmg/100 g body weight) � Fentanyl (analgesic; 0.01 mg/

100 g body weight). For left kidney harvesting, a single 10 minutes of warm ischemia, 10 minutes of reperfusion,and 40 minutes of warm ischemia (N � 8); (4) the Precdose of sodium heparin (1000 IU) was administered and

Torras et al: Ischemic preconditioning in the kidney2220

15/10 group had 15 minutes of warm ischemia, 10 minutes Design C. Finally, to determine whether precondition-ing affected cold ischemic damage, 21 rats were classifiedof reperfusion, and 40 minutes of warm ischemia (N �into three groups: (1) NoCOLD group, where kidneys8); and (5) the Prec 20/10 group had 20 minutes of warmwere flushed with EC solution at 4�C and immediatelyischemia, 10 minutes of reperfusion, and 40 minutes oftransplanted (N � 7); (2) COLD5H group, where kid-warm ischemia (N � 8).neys were flushed with EC at 4�C and stored for 5 hoursSecondly, given the optimum effect of 15 minutes ofbefore transplantation (N � 7); and (3) IP � COLD5Hwarm ischemic preconditioning, we tested the effects ofgroup, where after IP, the kidneys were flushed with ECincreasing the reperfusion time (20 and 40 min). To thisat 4�C and then stored for 5 hours before transplantationend, twelve animals were organized into two other(N � 7).groups: (1) the Prec 15/20 group had 15 minutes of warm

ischemia, 20 minutes of reperfusion, and 40 minutes ofTissue nitrite and nitrate measurementswarm ischemia (N � 6); and (2) the Prec 15/40 group

Nitric oxide production in renal tissue was determinedhad 15 minutes of warm ischemia, 40 minutes of reperfu-by tissue accumulation of nitrite and nitrate as describedsion, and 40 minutes of warm ischemia (N � 6).elsewhere [29]. Briefly, frozen kidney sections wereDesign B. Using the best schedule of preconditioningweighed and homogenized in 2 mL of 100 mmol/L Tris-from Design A, that is, 15 minutes of warm ischemiaHCl, pH 7.4 at 4�C. Proteins were precipitated in 1 mLfollowed by 10 minutes of reperfusion (Prec 15/10; here-of homogenate by adding 200 �L of 1 N HCl. Afterafter named IP), we determined whether NO could medi-centrifugation, the supernatant was adjusted to pH 7.6ate ischemic preconditioning. For this purpose, anotherwith 100 �L of 1 N NaOH plus 300 �L of 100 mmol/Lset of experiments was performed that included the fol-Tris-HCl. Total NO products were measured in the su-lowing groups: (1) ISC group with 40 minutes of warmpernatant with a commercial kit from Cayman Chemicalischemia (N � 9); (2) IP-ISC group, which had IP plus(Ann Arbor, MI, USA). Nitrate was reduced to nitrite40 minutes of warm ischemia (N � 14); and (3) NO-ISCby a three-hour incubation with nitrate reductase in thegroup administered spermine NONOate (10 mg/kg) IVpresence of nicotinamide adenine dinucleotide 3-phos-5 minutes before ischemia, and then 40 minutes of warmphate (NADPH) and flavin-adenine-dinucleotide. Ni-ischemia (N � 12).trite was converted into a deep purple azo compoundAdditional groups were studied to analyze the effectsby the addition of Griess reagent. Photometric measure-of blocking NOS on preconditioning: (1) NA-IP group,ment of the absorbance at 540 nm provided nitrite con-where the non-selective blocker L-NAME (10 mg/kg)centration. Protein concentration was determined in the

was added IV 5 minutes before surgery, then IP and other milliliter of homogenate following Bradford, withfinally 40 minutes of warm ischemia (N � 14); (2) NO- bovine serum albumin (BSA) as a standard. Results wereNA-IP group had spermine NONOate (10 mg/kg) and expressed as �mol NO/mg protein.the non-selective blocker L-NAME (10 mg/kg) adminis-tered IV 5 min before surgery, IP, and 40 minutes of Detection in situ and evaluation of DNA oxidationwarm ischemia (N � 9); (3) NA-ISC, where the non- To assess the involvement of free radical formationselective blocker L-NAME (10 mg/kg) was added IV in ischemia-reperfusion, we identified 8-oxo-2�-deoxy-5 minutes before surgery, and then 40 minutes of warm guanosine, a sensitive marker of DNA base damage, byischemia (N � 6); (4) AG-IP group, the selective induc- a modification of the Biotrin OxyDNA assay (BIOTRINible NOS blocker aminoguanidine (150 mg/kg) admin- Int Ltd, Dublin, Ireland). This test is based on an affinityistered subcutaneously (SC) one hour before surgery, technique that allows direct identification of DNA dam-IP, and 40 minutes of warm ischemia (N � 9); and (5) age in pathological specimens and in cultures in vitro [30].AG-ISC group, the selective inducible NOS blocker The OxyDNA assay applies a direct fluorescent tech-aminoguanidine (150 mg/kg) added SC one hour before nique for the detection of oxidative damage to DNA.surgery, 40 minutes of warm ischemia (N � 6). The re- Briefly, 5-�m thick tissue sections were pretreated withnal function in this set of experiments was monitored proteinase K (20 mg/mL) for 15 minutes. Then, the FITCfor only three days and no histological evaluation was conjugated with a specific oxyDNA probe, which bindsperformed. to 8-oxo-2�-deoxyguanosine in damaged cells, was added.

Finally, more animals were added to the Prec 15/10 All sections were examined under fluorescence micro-(N � 8), Prec 15/20 (N � 10), Prec 15/40 (N � 10), scope and semiquantitatively graded on a scale from 0NA-IP (N � 10) and AG-IP (N � 10) groups to assess to 3�, where 0 denotes no fluorescence, and 1, 2 and 3tissue NO and oxidized DNA. These animals followed mild, moderate and strong fluorescence, respectively.the same preconditioning schedule as described above

Statistical analysisexcept that the tissue was freeze-clamped instead ofapplying 40 minutes of warm ischemia. Kidneys from a The Chi square test was used to compare the mortality

ratio from acute renal failure between treated and non-Sham (N � 7) group were added.


Fig. 1. Assay of optimal one-cycle preconditioning schedule. Functional parameters. (A) A set of one-cycle schedules were used by occluding/releasing renal pedicles according to various warm ischemic (5, 10, 15, 20 min) and fixed reperfusion (10 min) windows, followed by 40 minutesof bilateral warm ischemia. Symbols and groups are: ISC (�), no preconditioning and 40 min of warm ischemia; (�) Prec 5/10, 5 min of warmischemia, 10 min of reperfusion, 40 min of warm ischemia; (�) Prec 10/10, 10 min of warm ischemia, 10 min of reperfusion, 40 min of warmischemia; (�) Prec 15/10, 15 min of warm ischemia, 10 min of reperfusion, 40 min of warm ischemia; and (�) Prec 20/10, 20 min of warm ischemia,10 min of reperfusion, 40 min of warm ischemia. Statistical analysis was by ANOVA and Scheffe’s test. a P � 0.05 ISC vs. Prec 5/10, Prec 10/10and Prec 15/10; b P � 0.05 ISC vs. the other groups. (B) A set of one-cycle schedules with fix ischemic (15 min) and various reperfusion windows(10, 20, 40 min), followed by 40 min of bilateral warm ischemia. Symbols and groups are: (�) ISC; (�) Prec 15/10; (�) Prec 15/20, 15 min ofwarm ischemia, 20 min of reperfusion, 40 min of warm ischemia; and (�) Prec 15/40, 15 min of warm ischemia, 40 min of reperfusion, 40 min ofwarm ischemia. Statistical analysis was by ANOVA and Scheffe’s test. a P � 0.05 Prec 15 to 10 vs. ISC, b P � 0.05 Prec 15/10 vs. Prec 15/20 andPrec 15/40.

treated groups. To compare more than two groups for similar to that in ISC rats. All ischemic preconditioningproteinuria and serum creatinine throughout the follow- groups showed progressive amelioration of renal func-up, a one-way analysis of variance (ANOVA) followed tion on the second and third days after ischemia. Serumby Scheffe’s test was performed. To compare histologi- creatinine levels in the Prec 5/10, Prec 10/10 and Preccal data, the non-parametric Kruskal-Wallis test and sub- 15/10 groups returned to basal values, whereas that ofsequent Connover’s test were applied. All P values were the Prec 20/10 did not. Among preconditioning groups,two-tailed, and a P value of less than 0.05 was consid- rats with 15 minutes of ischemic preconditioning (Precered statistically significant. Data are the mean � stan- 15/10) had the best renal function on the first and seconddard error. days after ischemia.

Histological evaluation revealed severe tubulointersti-tial damage in kidneys from the ISC group, while onlyRESULTSthe Prec 15/10 group had a significantly lower degreeProtective effect of preconditioning onof histological damage, as evidenced by lower tubularwarm ischemia to establish the optimalnecrosis, medullar congestion and hemorrhage, and de-one-cycle preconditioning schedulesvelopment of proteinaceous casts (Table 1). The otherThe serum creatinine (SCr) profile is shown in Fig-preconditioning groups showed tubulointerstitial dam-ure 1A. Rats from the ISC group presented severe renalage similar to that of ISC group.failure on the first day and serum creatinine worsened

In the groups with the reperfusion window prolongeduntil a peak on the second day. On the seventh dayto 20 or 40 minutes (Prec 15/20 and Prec 15/40 groups)these animals showed a SCr concentration higher thanthe SCr was higher than in Prec 15/10 animals from thethe baseline value. On the first day, serum creatinine infirst day. This difference reached significance on the sec-the Prec 5/10, Prec 10/10 and Prec 15/10 groups wasond and third days (Fig. 1B). Tubulointerstitial damagesignificantly lower than in ISC rats. In contrast, the 20/10in these groups was clearly stronger than in Prec 15/10preconditioning schedule did not protect against ische-

mia since the SCr profile in the Prec 20/10 group was animals and similar to that of the ISC group (Table 1).


Table 1. Assay of optimal one-cycle preconditioning schedule NO after the preconditioning 15/10 window, to a valuesimilar to that found in Sham animals.Score

ISC 2.9�0.4Detection in situ and evaluation of DNA oxidationPrec 5/10 2.1�0.1

Prec 10/10 2.0�0.2 The extent of DNA oxidation, as measured by tissuePrec 15/10 1.7�0.2a

8-oxo-2�-deoxyguanosine, showed a profile parallel toPrec 20/10 2.6�0.5Prec 15/20 2.9�0.4 that of nitric oxide with a higher fluorescence intensityPrec 15/40 2.8�0.3 in the group of the preconditioning 15/10 window thanP 0.035

in sham-operated rats (Figs. 2 and 3). As reperfusion wasAbbreviations are in the text. Samples were examined for tubular cell necrosis, prolonged, both the intensity and number of positivetubular dilation, intratubular cell detachment, interstitial edema and interstitial

cellular infiltrate. Abnormalities were graded from 0 to 4� (0 � no abnormalities; cells increased, suggesting higher DNA oxidation by oxy-1� � changes affecting �25% of the sample; 2� � changes affecting 25 to 50% gen free radicals.of the sample; 3� � changes affecting 50 to 75% of the sample; and 4� �changes affecting �75% of the sample). Statistical analysis was by the Kruskal-Wallis/Connover’s test. Protective effect of preconditioning on cold ischemia

a P � 0.05 vs. all the other groupsAll animals survived the study period. Over the seven-

day follow-up, SCr in NoCOLD group was similar to pre-operative values. Animals grafted after five hours of cold

Effect of NO donor on warm ischemia and effect of ischemia (COLD5H group) had significantly higher SCr

NOS inhibition on preconditioning levels than NoCOLD animals throughout the study. Onthe first day, SCr in IP � COLD5H increased as in theNitric oxide addition with a direct NO donor to ani-COLD5H group. However, on the following days itmals subjected to 40 minutes of warm ischemia decreasedsteadily decreased to values significantly lower than inSCr to levels even lower than in preconditioned animalsCOLD5H and similar to those found in NoCOLD ani-(Table 2). This indicates that NO donors provide signifi-mals (Fig. 4).cant protection against dysfunction resulting from warm

The histological evaluation was consistent with func-renal ischemia.tional results. The non-ischemic group (NoCOLD) hadWhen NOS was inhibited non-selectively withscarcely appreciable lesions, whereas the COLD5H groupL-NAME, the effect of preconditioning was abolished,displayed moderate-severe ischemic-characteristic tubu-and severe renal failure was observed with SCr notablylointerstitial lesions. The IP � COLD5H group showedhigher than in ISC animals (Table 2). Furthermore, 6 ofsome ischemic tubulointerstitial abnormalities, which were14 animals (43%) from this NA-IP group died becauseclearly slighter than in the COLD5H group (Table 3 andof uremia, suggesting that the ischemic damage was alsoFig. 5).greater. Accordingly, in the group of non-preconditioned

animals receiving L-NAME (NA-ISC group), compara-ble high mortality rates (33%, 2 of 6) and severe renal DISCUSSIONfailure were observed (Table 2). When we added the Preconditioning is a simple and harmless method usedNO donor to the group of preconditioned animals receiv- to render an organ tolerant to ischemia. Here, we exam-ing L-NAME (NO-NA-IP group), SCr fell to levels similar ined whether a one-cycle schedule of preconditioningto those found in preconditioned animals. attenuated the damage induced by sustained ischemia

Finally, selective blocking of inducible NOS with ami- in the kidney and then determined the optimal timenoguanidine in preconditioned animals (AG-IP group) window of ischemia and reperfusion. The main findingabolished ischemic preconditioning and SCr increased to is that a transient and short episode of warm ischemialevels similar to those in ISC animals. Accordingly, the and reperfusion protects the kidney from subsequentgroup of non-preconditioned animals receiving amino- warm or cold ischemia insults by a mechanism in whichguanidine (AG-ISC group) presented a SCr profile similar NO is involved.to that of ISC animals. Neither group showed mortality. In the warm ischemia setting, one-cycle of 15 minutes

of ischemia followed by 10 minutes of reperfusion wasNitric oxide content in preconditioned tissue the optimal time schedule, as it prevented both renal

Tissue NO, as measured by nitrite and nitrate levels, function and morphology. Alternatively, we showed thatwas significantly higher after 15 minutes of warm ische- either 5 or 10 minutes of ischemia followed by 10 minutesmia and 10 minutes of reperfusion, the preconditioning of reperfusion offered functional but partial structural15/10 window, than in sham-operated rats (Fig. 2). When protection. However, when the ischemic window wasreperfusion was prolonged to 20 and 40 minutes, nitrite prolonged beyond 15 minutes, the preconditioning effectand nitrate levels increased significantly. Both the non- disappeared. Previous studies on warm renal ischemia

[12, 13] used a four-cycle preconditioning schedule, whichselective and inducible NOS blocking decreased tissue


Table 2. Effects of niric oxide (NO) donor and NO synthase (NOS) inhibition on ischemic preconditioning

Prec NO NOS15-10 donor inhibitor SCr 0 SCr 1 SCr 2 SCr 3

ISC — — — 45 �2 273 �20abc 274 �43abc 228�49abc

IP-ISC Yes — — 46 �2 183 �25 121�20 83�9NO-ISC — Yes — 44�3 103 �11ac 74�4 66�3NA-ISC — — L-NAME 46�2 268 �10abc 227 �28abc 157�3abc

NA-IP Yes — L-NAME 45�1 327 �14abc 409 �43abcd 289�49abc

NO-NA-IP Yes Yes L-NAME 45�2 191 �32 122�24 86�15AG-ISC — — AG 46�2 263 �21abc 318 �44abc 276�44abc

AG-IP Yes — AG 44�2 295 �17abc 295 �52abc 221�40abc

P NS 0.0001 0.0001 0.0001

Using the 15/10 preconditioning schedule, several groups were studied using either a direct NO donor, spermine NONOate (NO, 10 mg/kg), or two NOS blockers(L-NAME 10 mg/kg and aminoguanidine, AG 150 mg/kg). Definitions of groups are: ISC group, 40 min of warm ischemia; IP-ISC group, preconditioning 15/10,40 min of warm ischemia; NO-ISC group, spermine NONOate, 40 min of warm ischemia; NA-IP group, the non-selective blocker L-NAME, preconditioning 15/10,40 min of warm ischemia; NO-NA-IP group, spermine NONOate, L-NAME, preconditioning 15/10, 40 min of warm ischemia; NA-ISC, L-NAME, 40 min of warmischemia; AG-IP group, the selective inducible NOS blocker aminoguianidine, preconditioning 15/10, 40 min of warm ischemia; and AG-ISC group, aminoguanidine,40 min of warm ischemia. In the NA-IP and NA-ISC groups, 43 and 33% mortality was observed, respectively, because of uremia. Statistical analysis is by ANOVA/Scheffe’s test.

a P � 0.05 vs. IPb P � 0.05 vs. NO-ISCc P � 0.05 vs. NO-NA-IPd P � 0.05 vs. ISC, AG-ISC and AG-IP

Fig. 2. Tissue nitric oxide and DNA oxida-tion after one cycle preconditioning. (A) Nitricoxide (NO) production in renal tissue was de-termined by tissue accumulation of nitrite andnitrate. DNA oxidative damage was evaluatedby a direct fluorescent technique, identifying8-oxo-2�-deoxyguanosine. (B) All sections wereexamined with fluorescence microscopy andsemiquantitatively graded from 0 to 3�, where0 represents no fluorescence intensity, and 1,2, 3, mild, moderate and strong fluorescence in-tensity respectively. Statistical analysis was bythe Kruskal-Wallis/Connover test; *P � 0.05.

is the most widespread method used in experimental cedure of preconditioning. However, a previous studywith a four-cycle schedule, in which the reperfusion timemodels [11, 15]. However, this procedure may be confus-

ing and complex, and results have not always been con- was further increased by 30 minutes, disclosed a similarlack of protection against renal ischemia [12]. These find-clusive in the kidney. The efficacy of our method with

only one cycle of ischemia and reperfusion offers further ings conflict with heart studies in which preconditioninginduces not only an early protective interval that may ex-advantages and brings preconditioning closer to the clin-

ics. In fact, this one-cycle preconditioning schedule has tend to one hour, but also a second window of protectionat 24 hours after preconditioning [11]. Thus, our resultsbeen reported by other groups to be effective in the liver

[5, 6] and the small bowel [8], describing protection on suggest that the interval of protection induced by precon-ditioning varies according to the organ, and is extremelythe injury associated with ischemia of the organ.

Most of the studies with one-cycle preconditioning short in the kidney.Nitric oxide has been linked to ischemic precondi-schedules only assess ischemic windows while they keep

reperfusion unchanged [5, 6]. Our current study also tioning in several organs. To date, in the kidney NO hasbeen associated only with the mechanisms of ischemicevaluated the reperfusion window, showing that when it

was prolonged to 20 or 40 minutes following 15 minutes damage [22, 26] but its role as innate cellular protectorin preconditioning has been scarcely reported. Thus, aof ischemia, the preconditioning effect disappeared. It

can be argued that this one-cycle schedule is a weak pro- rapid generation of renal NO has been evidenced after

Fig. 3. In situ detection of DNA oxidation. Kidneys from warm isch-emic study were marked for 8-oxo-2�-deoxyguanosine and examinedwith fluorescence microscopy. Representative images (20) are fromsham-operated (A), Prec 15/10 (B), and Prec 15/40 (C ) groups.

Fig. 5. Effect of preconditioning on cold ischemia. Kidneys from thecold ischemic study were evaluated for conventional histology. Repre-sentative images (PAS, 20) are from NoCOLD (A), COLD5H (B),and IP � COLD5H (C ) groups are shown.


Table 3. Effects of preconditioning on cold ischemia

Score

NoCOLD 0.1�0.0COLD5H 2.0�0.3a

IP � COLD5H 0.6�0.2P 0.0016

Histological evaluation was performed similarly to warm renal studies. Statisti-cal analysis was by Kruskal-Wallis/Connover’s test.

a P � 0.05 COLD5H vs. No COLD and IP � COLD5H

Despite the higher production of renal nitrite and ni-trate levels when reperfusion was prolonged to 20 or 40minutes, the preconditioning effect was not observed.This suggests that the protective window induced byendogenous NO is notably narrow. We thus aimed toelucidate these results. During reperfusion, tissue 8-oxo-2�-deoxyguanosine, a highly sensitive cellular marker of

Fig. 4. Effect of preconditioning on cold ischemia. Functional parame- oxidative damage [26], increased with renal nitrite andters after the 15/10 preconditioning schedule are shown in kidneys nitrate levels. Although free radical production is ancold-stored for 5 hours in Euro-Collins (EC) solution and transplanted

early and relatively transient event [31], as reperfusionsyngeneically. Symbols and groups are: (�) NoCOLD, kidneys flushedwith EC solution at 4�C and immediately transplanted; (�) COLD5H, damage advances, tissue structures, including DNA, be-kidneys flushed with EC at 4�C and stored for 5 hours before trans- come progressively oxidized [32]. The higher tissueplantation; and (�) IP � COLD5H, preconditioning 15/10, kidneys

8-oxo-2�-deoxyguanosine levels in Prec 15/20 and Precflushed with EC at 4�C and stored for 5 hours before transplantation.Statistical analysis is by ANOVA/Scheffe’s test. a P � 0.05 NoCOLD 15/40 groups reflect the presence of a bulk of free radicalsvs. COLD5H; b P � 0.05 NoCOLD vs. IP-COLD5H; c P � 0.05 IP � that may oxidize NO and thus inactivate its physiologicalCOLD5H vs. COLD5H.

protective effect. These higher NO levels may thus be-come cytotoxic, presumably owing to peroxynitrite for-mation. Furthermore, although a low level of NO mayup-regulate anti-apoptosis through protecting moleculesvarious periods of warm ischemia (15 to 45 min), whichsuch as Bcl-2 and Bcl-xl [33], the interaction betweenfurther increased as reperfusion was prolonged [26]. Al-free NO and superoxide initiates an apoptotic process [34]though short- and long-term ischemia induced similarthat aggravates ischemic damage. Similar approaches haveNO production, the former caused lower free radicalbeen reported by studies on hepatic preconditioning [6],production and histological damage. Our results revealbut instead of reperfusion, which was kept constant, Per-a similar comportment, since renal nitrite and nitratealta et al tested various intervals of warm ischemia. In thelevels increased at the end of the protective one-cycleliver, the optimal preconditioning window was defined bypreconditioning schedule. Furthermore, as only moder-the balance between the tissue concentration of adeno-ate tissue 8-oxo-2�-deoxyguanosine was observed at thissine-NO and xanthine produced during ischemia. There-moment, NO may exert its optimal physiological func-fore, in our one-cycle preconditioning schedule, the dele-

tions with little interference by free radicals. Thus, theterious effect of oxidative damage induced by ischemia

generation of NO when subsequent prolonged warm prevails over the beneficial effect of endogenous NOischemia is produced may account for the beneficial ef- release when reperfusion lasts more than 10 minutes.fect of renal preconditioning. The protective effect of Pharmacological manipulation with the non-selectivethe exogenous addition of the NO donor, reproducing NOS blocker also supports the implication of NO inischemic preconditioning, clearly supports this hypothe- renal ischemic preconditioning. First, renal nitrite andsis. The results achieved with this maneuver agree with nitrate levels were reduced after L-NAME adminis-a heart study in which exogenous NO triggered a precon- tration. Second, renal function was not protected, thusditioning state, although controversial results were re- indicating that the preconditioning effect had been abol-ported when endogenous NO was induced by precondi- ished. And third, the addition of the NO donor to pre-tioning [23]. In this way, low doses of NO donor in the conditioned animals treated with L-NAME restored theheart induce the rapid expression of two antioxidant and preconditioning effect. As reported elsewhere [26], theanti-apoptotic proteins, heat shock protein 70 (HSP70) use of this non-selective NOS blocker further impairedand hemeoxygenase-1 (HO-1), which attenuate the sub- renal damage and resulted in high mortality, thus con-

firming that the NO system is a natural defense of cellssequent ischemia/reperfusion injury [21].


against ischemic injury. Preconditioning studies in other short cold ischemia time, previous studies showed thatit caused severe acute renal failure [27]. In clinical renalorgans have also shown that NO production is stimulated

by endogenous adenosine [14, 20]. During a short isch- harvesting, potential protective maneuvers should be assimple as possible. The one-cycle schedule proposed hereemia period, there is accumulation of adenosine from

adenosine 5�-triphosphate (ATP) degradation and dur- thus might be an appropriate procedure in humans. Asthe ischemia and, especially, reperfusion windows pre-ing reperfusion NO is generated as a result of adenosine

receptor activation [4]. In the kidney, adenosine and sented are brief enough, ischemic preconditioning maybe a suitable procedure in clinical renal transplantation.adenosine agonists may contribute to the attenuation of

ischemia-reperfusion injury through A1 or A2A receptor In summary, ischemic preconditioning improves theacute renal failure induced by warm and cold ischemia-activation [14, 35] or A3 antagonism [14].

Since the selective blockade of inducible NOS (iNOS) reperfusion injury. In the rat kidney, 15 minutes of warmischemia and 10 minutes of reperfusion is the optimalproduced not only the early inhibition of nitrite and

nitrate generation but also the disappearance of the pre- schedule for preconditioning. Our data indicate that theprotective effect of renal ischemic preconditioning is re-conditioning effect, we aimed to determine whether this

enzyme is involved in this early phase of ischemic precon- lated to the local production of NO and that the effectmay be lost depending on the balance between NO andditioning. This may be a controversial issue, since the

synthesis de novo of iNOS may take longer than the oxidative stress induced by preconditioning. This endog-enous protective mechanism may be promising in clinicaltime included in our schedule. Previous experiments

have shown that ischemic preconditioning is associated renal preservation.with rapid activation of tyrosine kinases [36, 37] and thatiNOS undergoes a post-translational modification via ty- ACKNOWLEDGMENTSrosine phosphorylation, resulting in enzymatic activation This study was supported by a grant from Fondo de Investigaciones

Sanitarias (98/0029-02). Immaculada Herrero-Fresneda is a post docas early as 5 minutes after experimental manipulationfellow from Instituto de Salud Carlos III (BISCIII 99/4262), Marta[38]. Therefore, the iNOS tonically present in the kidneyRiera Oliva from Fondos FEDER Plan Nacional I � D (2FD1997-2109-

may have been activated by preconditioning and conse- C02-01) and Nuria Lloberas from “Fundacio August Pi Sunyer.” Partof this study was presented at the XXXVII Congress of the EDTA,quently offer renal protection. As the specific blocker(Nice, September 2000) and at the XVIII International Congress ofwas administered early enough to ensure its maximalthe Transplantation Society (Rome, August 2000).

action, although we did not measure transcription ortranslation of iNOS, the lowering in renal nitrite and Reprint requests to Josep M. Grinyo, M.D., Nephrology Department,

Hospital of Bellvitge, CSUB, Feixa Llarga s/n, E-08907, L’Hospitalet,nitrate levels is probably due to a reduction in NO byBarcelona, Spain.

this pathway. The dose of aminoguanidine used in our E-mail: [email protected] may have inhibited not only iNOS, but also endo-thelial NOS (eNOS), as the compound is only 10- to REFERENCES40-fold selective for iNOS versus eNOS [24]. However,

1. Land W, Messmer K: The impact of ischemia/reperfusion injuryto our knowledge all the pharmacological iNOS blockers on specific and non-specific early and late chronic events afteravailable are poorly selective or have effects on eNOS. organ transplantation. Transplant Rev 10:108–127, 1996

2. Moreso F, Gallen M, Garcia-Osuna R, et al: Multivariate analy-Thus, although our results suggest the participation ofsis of prognostic factors in renal transplantation. Transplant ProciNOS in early renal preconditioning, further experi- 27:2226–2228, 1995

ments, like the use of knockout mice for iNOS or anti- 3. Meldrum D, Mitchel MB, Banerjee A, Harken AH: Cardiacpreconditioning can trigger a preconditioned state through a freesense oligodeoxynucleotides, should be performed andradical mechanism, but endogenous nitric oxide is not a trigger ofcarefully discussed. classical ischemic preconditioning. J Mol Cell Cardiol 32:1159–

Ischemic preconditioning was first described as a 1167, 20004. Parrat J: Protection of the heart by ischemic preconditioning:method to protect the heart from myocardial infarction

Mechanisms and possibilities for pharmacological exploitation.when sustained ischemia is induced either in minimally Trends Pharmacol Sci 15:19–25, 1994invasive percutaneous angioplasty or during anastomosis 5. Peralta C, Hotter G, Closa D, et al: J. Protective effect of precon-

ditioning on the injury associated to hepatic ischemia-reperfusionof the vascular graft [39]. In other organs, precondition-in the rat: Role of nitric oxide and adenosine. Hepatology 25:934–ing may be used to study not only innate defenses against937, 1997

ischemic injury but also its great potential for organ 6. Peralta C, Closa D, Xaus C, et al: Hepatic preconditioning inrats is defined by a balance of adenosine and xanthine. Hepatologypreservation [40, 41]. Thus, we evaluated the effect of28:768–773, 1998our one-cycle schedule on cold ischemia in rat renal

7. Osborne DL, Aw TY, Cepinskas G, Kvietys PR: Developmenttransplantation, keeping in mind it’s potential for renal of ischemia/reperfusion tolerance in the rat small intestine. An

epithelium-independent event. J Clin Invest 94:1910–1918, 1994preconditioning in clinical transplantation. Precondi-8. Hotter G, Closa D, Prados M, et al: Intestinal preconditioningtioning improved renal function during the seven-day

is mediated by a transient increase in nitric oxide. Biochem Biophysfollow-up and, more importantly, the renal structure was Res Commun 222:27–32, 1996

9. Heurteaux C, Lauritzen I, Widmann C, Lazdunski M: Essentialalso preserved. Although five hours of preservation is a


role of adenosine, adenosine A1 receptors, and ATP-sensitive K� superoxide and peroxynitrite formation. Biochem Soc Trans 21:330–334, 1993channels in cerebral ischemic preconditioning. Pharmacology 92:

26. Weight SC, Furness PN, Nicholson ML: Nitric oxide generation4666–4670, 1996is increased in experimental renal warm ischaemia-reperfusion in-10. Yoshioka T, Bills T, Moore-Jarret T, et al: Role of intrinsicjury. Br J Surg 85:1663–1668, 1998antioxidant enzymes in renal oxidant injury. Kidney Int 38:282–

27. Herrero-Fresneda I, Torras J, Riera M, et al: Prevention of cold291, 1990ischemia-reperfusion injury by an endothelin receptor antagonist11. Bolli R: The late phase of preconditioning. Circ Res 87:972–in experimental renal transplantation. Nephrol Dial Transplant 14:983, 2000872–880, 199912. Islam C, Mathie R, Dinneeen MD, et al: Ischemia-reperfusion

28. Herrero-Fresneda I, Torras J, Lloberas N, et al: Cold ischemiainjury in the kidney; the effect of preconditioning. BJU Int 79:842–in the absence of alloreactivity induces chronic transplant nephrop-847, 1997athy through a process mediated by the platelet-activating factor.13. Jefayri MK, Grace PA, Mathie RT: Attenuation of reperfusionTransplantation 70:1624–1631, 2000injury by renal ischemic preconditioning: The role of nitric oxide.

29. Hortelano S, Genaro A, Bosca L: Phorbol esters induce nitricBJU Int 85:1007–1013, 2000oxide synthase activity in rat hepatocytes. J Biol Chem 267:24937–14. Lee HT, Emala CW: Protective effects of renal ischemic precondi-24940, 1992tioning and adenosine pretreatment: Role of A1 and A3 receptors.

30. De Zwart L, Meerman H, Commandeur J, Vermeulen N: Bio-Am J Physiol (Renal Physiol) 278:F380–F387, 2000markers of free radical damage, applications in experimental ani-15. Bolli R, Manchikalapudi S, Tang XL, et al: The protective ef-mals and in humans. Free Radic Med 26:202–226, 1999fect of late preconditioning against myocardial stunning in con-

31. Lloberas N, Torras J, Herrero I, et al: The ether phospholipidsscious rabbits is mediated by nitric oxide synthase. Circ Res 81: trail: Blood timing in renal ischemia-reperfusion injury. Transplant1094–1101, 1997 Proc 34:47–48, 2002

16. Riera M, Herrero I, Torras J, et al: Ischemic preconditioning 32. Szabo C, Ohshima H: DNA damage induced by peroxynitrite:improves post-ischemic acute renal failure. Transplant Proc 31: Subsequent biological effect. Nitric Oxide 1:373–385, 19972346–2347, 1999 33. Genaro AM, Hortelano S, Alvarez A, et al: Splenic B lympho-

17. Marber MS, Latchman DS, Walker JM, Yellon DM: Cardiac cyte programmed cell death is prevented by nitric oxide releasestress protein elevation 24 hours after brief ischemia or heat stress through mechanisms involving sustained Bcl-2 levels. J Clin Investis associated with resistance to myocardial infarction. Circulation 95:1984–1999, 199588:1264–1272, 1993 34. Okusa M, Linden J, MacDonald T, Huang L. Selective A2a

18. Mizushima Y, Wang P, Jarrar D, et al: Preinduction of heat shock adenosine receptor activation reduces ischemia-reperfusion injuryproteins protects cardiac and hepatic functions following trauma in rat kidney. Am J Physiol (Renal Physiol) 277:F404–F412, 1999

35. Baines CP, Wang L, Cohen MV, Downey JM: Protein tyrosineand hemorrhage. Am J Physiol 278:R352–R359, 2000kinase is downstream of protein kinase C for ischemic precondi-19. Peralta C, Closa D, Hotter E, et al: Liver ischemic precondi-tioning’s anti-infarct effect in the rabbit heart. J Mol Cell Cardioltioning is mediated by the inhibitory action of nitric oxide on30:383–392, 1998endothelin. Biochem Biophys Res Com 229:264–270, 1996

36. Sandau K, Pfelishifter J, Brune B: The balance between nitric20. Peralta C, Hotter G, Closa D, et al: The protective role ofoxide and superoxide determines apoptotic and necrotic cell deathadenosine in inducing nitric oxide synthesis in rat liver ischemiaof rat mesangial cells. J Immunol 158:4938–4949, 1997is mediated by activation of adenosine A2 receptors. Hepatology

37. Ping P, Zhang J, Zheng YT, et al: Demonstration of selective29:126–132, 1999protein kinase C-dependent activation of Src and Lck tyrosine21. Katori M, Tamaki T, Takahashi T, et al: Prior induction of heatkinases during ischemic preconditioning in conscious rabbits. Circshock proteins by a nitric oxide donor attenuates cardiac ischemia/Res 85:542–550, 1999reperfusion injury in the rat. Transplantation 69:2530–2537, 2000

38. Pan J, Burgher KL, Szczepanik AM, Ringheim GE: Tyro-22. Garcia-Criado G, Eleno N, Santos-Benito F, et al: Protective sine phosphorylation of inducible nitric oxide synthase: Implica-effect of exogenous nitric oxide on the renal function and inflam- tions for potential post-translational regulation. Biochem J 314:matory response in a model of ischemia-reperfusion. Transplanta- 889–894, 1996tion 27:982–990, 1998 39. Murry CE, Jennings RB, Reimer K: Preconditioning with ische-

23. Nakano A, Liu GS, Heusch G, et al: Exogenous nitric oxide can mia. A delay of lethal cellular injury in ischemic myocardium.trigger a preconditioned state through a free radical mechanism, Circulation 74:1124–1136, 1986but endogenous nitric oxide is not a trigger of classical ischemic 40. Saitoh Y, Gu K, Kin S, et al: Ischemic preconditioning improvespreconditioning. J Mol Cell Cardiol 32:1159–1167, 2000 cardiac functional recovery following preservation with University

24. Southan GJ, Szabo C: Selective pharmacological inhibition of of Wisconsin solution. Transplantation 60:1079–1086, 1995distinct nitric oxide synthase isoforms. Biochem Pharmacol 51:383– 41. Yin DP, Sankary HN, Chong ASF, et al: Protective effect of394, 1996 ischemic preconditioning on liver preservation-reperfusion injury

in rats. Transplantation 66:152–157, 199825. Beckman JS, Crow JP: Pathological implications of nitric oxide,

promising effects of ischemic preconditioning in renal transplantation

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