Vasodilating Prostaglandins Attenuate IschemicRenal Injury Only if Thromboxane is Inhibited

JOSEPH M. KLAUSNER, M.D., IAN S. PATERSON, M.B., F.R.C.S., LESTER KOBZIK, M.D., CYNTHIA RODZEN, B.S.,C.R. VALERI, M.D., DAVID SHEPRO, PH.D., and HERBERT B. HECHTMAN, M.D.

Ischemia-induced renal injury is prevented by inhibition ofthromboxane (Tx) synthesis. This protection was believed to besecondary to a high prostaglandin (PG)/TxA2 ratio. This studytests whether increasing the PG/Tx ratio by administration ofvasodilating PGs protects the reperfused ischemic kidney.Anesthetized rats underwent right nephrectomy and 45 minutesof left renal pedicle clamping.B ing 10 minutes before clamprelease, animals were treated intravenously with the following:saline placebo (n = 10); the cyclooxygenase inhibitor ibuprofen(Ibu), 12.5 mg/Kg in a bolus (n = 8); a stable analogue of pros-tacyclin (PGI2), 500 ng/kg/minute for 2 hours (n = 9); PGEI,400 ng/kg/minute for 2 hours (n = 8); the combination Ibu andPGI2 (n = 8) or PGE, (n = 8). In saline treated ischemic controls,5 minutes after reperfusion plasma, thromboxane (TxB2) and 6-keto-PGF, levels were 2537 and 317 pg/ml, respectively-higherthan the TxB2 and 6-keto-PGF1 levels of 750 and 80 pg/ml,respectively, in nephrectomized but nonischemic sham controls(n = 7) (p < 0.05). In ischemic control animals at 24 hours,creatinine levels were 4.6 mg/dl, relative to 0.9 ml/dl in shamanimals (p < 0.05); the weight of the left (L) ischemic kidneyrelative to the right (R) normal kidney was 118%, comparedwith 99% in sham animals (p < 0.05); and renal histology ofischemic control animals at 24 hours showed acute tubular ne-crosis (ATN) relative to normal findings in sham animals. Pre-treatment with Ibu led to: TxB2 and 6-keto-PGF, levels of 116and 40 pg/ml, lower than those of sham animals (p < 0.05);creatinine levels of 4.6 mg/dl, L/R renal weight of 119%; andATN similar to that of ischemic controls. Treatment with a PGI2analogue or PGE, was not protective and led to increases inTxB2, 6-keto-PGF1, creatinine, L/R renal weight, and ATNsimilar to that of ischemic controls. The combination of Ibu andeither PGI2 or PGE, led to: reduced levels of TxB2 and 6-keto-PGF, (p < 0.05); attenuated increases in creatinine to 2.2 and2.3 mg/dl, respectively (p < 0.05); and limited ATN (p <0.05).These data indicate that the vasodilating PG protect the ischemicreperfused kidney only when Tx is inhibited.

Supported in part by The National Institute of Health, Grants No.GM24891-10, HL16714-03; The U.S. Navy Office of Naval Research,Contract No. N00014-79-C-0168; The Brigham Surgical Group, Inc.;and The Trauma Research Foundation.

Reprint requests and correspondence: Herbert B. Hechtman, M.D.,Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.

Submitted for publication: June 16, 1988.

From the Department of Surgery, Brigham and Women'sHospital, and Harvard Medical School, The Naval Blood

Research Laboratory, Boston University School of Medicine,and The Biological Science Center, Boston University,

Boston, Massachusetts

I SCHEMIA INDUCED RENAL INJURY is associated withincreased levels of prostacyclin (PGI2) and throm-boxane (Tx)B2 in renal vein plasma." 2 Previous work

has indicated that the PGI2/TxA2 ratio is a determinantofischemic injury. 1,2 Thus, pretreatment with the Tx syn-thetase inhibitor OKY 046 that increases the ratio, butnot with a cyclooxygenase inhibitor that decreases theratio, prevents acute tubular necrosis (ATN). Further, thevariable protection offered by Tx inhibitors appears to bebased on their ability both to lower Tx synthesis and en-hance PGI2 production by redirection of precursor en-doperoxides.3 Based on these data, it was postulated thatexogenous administration of vasodilating PG would in-crease the PG/Tx ratio and thereby protect the ischemickidney. Previous studies with vasodilating PGs in modelsof acute renal failure in which blood flow was abolishedor markedly reduced are inconclusive. Although PG12 orPGE2 have yielded no protection in several studies,4 inothers, some benefit in using the same compounds havebeen gained.5'6 Thus, PGs of the "I" and "E" series (e.g.,PGI2, PGE,) attenuated the histologic changes of ATNafter a renal ischemic insult in dogs, but blood chemistrydata did not support improvement in renal function.5 Alow dose ofPGI2 attenuated the reduced inulin clearanceand ATN after 40 minutes of renal ischemia in volume-loaded rats, but this protection could have been due largelyto the volume expansion induced by inulin infusion.6 Thisstudy tests the ability of vasodilating PGs to moderateischemia-induced renal failure in our well-defined ratmodel.

219

301 RENAL VEIN rxaB analogue nor PGE, are metabolized to 6-keto-PGF1 andtherefore do not contribute to this prostanoid end-product.After 45 minutes of renal ischemia, the abdomen was

* opened and the vascular clamp was released. Five minuteslater, a 2-ml blood sample was drawn from the left renal

2000 - vein for measurement of prostanoids, and the laparotomywas sutured closed. The vascular catheters were removedat the end ofthe 2-hour drug infusion. Twenty-four hoursafter ischemia, the rats were reanesthetized, and 3 ml ofblood was drawn from the left renal vein for assay of1000 _ creatinine. The left kidney was then removed. Its weightwas compared with that of the previously removed rightkidney and expressed as a percentage. The kidney wasthen fixed for histologic evaluation. Finally, the rats were

011 mm * t _1killed with potassium chloride administered intrave-0f. S nously.4/' <g,° / g t < tProstanoids were assayed in blood drawn in syringes

containing 0.2 ml of 0.07 M ethylene diamine tetraceticFMG. 1. After 5 minutes of reperfusion, renal ischemia led to high renal acid and 0.09 M aspirin. The blood was immediately cen-vein TxB2 levels in saline-placebo-treated animals, relative to sham con- trifuged and the plasma frozen at -20 C. Plasma TxB2trols. Ibu alone or combined with the PG12 analogue or PGE, led to low and 6-keto-PGFI, the stable hydrolysis products of TxA2TxB2 levels and prevented the ischemia-induced rise in TxB2. Either the and PGI2, were measured by radioimmunoassay.7 Cre-PG!2 analogue or PGE, alone led to high TxB2 levels, similar to thoseof placebo ischemic controls. Asterisks refer to significant differences atinine levels were estimated colorimetrically after the ad-relative to sham animals, and daggers indicate significant differences dition of picric acid.8 Histologic sections of the kidneybetween placebo- and drug-treated rats. were evaluated in a "blinded" fashion. Each kidney was

graded for the extent of ATN based on the percentageMethods involved by ATN in the corticomedullary junction areaSixty adult male, Sprague-Dawley rats (Charles River ofa midsagittal section. A grading system of0-4 was used

Lab, Wilmington, MA) weighing approxinately 500 g were where 0 represented a normal kidney; 1 = minimal ATN,anesthetized with 60 mg/kg pentobarbitol administered less than 5%; 2 = mild, 5-25% ATN; 3 = moderate, 25-intraperitoneally. A jugular venous catheter was inserted 75% ATN; and 4 = severe, more than 75% ATN.in all animals for fluid and drug administration, and in Results are reported as the mean ± standard error. Sta-18 animals (two to four rats in each group), the carotid tistics were performed using an analysis of variance andartery was also cannulated for arterial pressure monitor- a nonpaired t-test; p <0.05 was considered statisticallying. A right nephrectomy was performed through a mid- significant.line laparotomy. The left renal pedicle was then isolated Animals used in this study were maintained in accor-and occluded for 45 minutes with a vascular clamp. In dance with the guidelines of the Committee on Animalssham animals, after right nephrectomy, the left renal ped- of the Harvard Medical School and those prepared by theicle was encircled but not occluded. The abdominal in- Committee on Care and use of Laboratory Animals ofcision was then closed loosely. Beginning 10 minutesbe- the Institute of Laboratory Animal Resourses, Nationalfore clamp release and continuing for 2 hours, all animals Research Council.were treated intravenously at an infusion rate of 1.9 ml/

Resultshour with either saline alone (n = 10), or saline in com-bination with the following drugs: ibuprofen (Ibu) (Up- After 45 minutes of renal ischemia and 5 minutes ofjohn Company, Kalamazoo, MI) 12.5 mg/kg as a bolus; reperfusion, TxB2 levels in renal vein plasma of saline-R0236416,Tm a stable fluorinated PGI2 analogue (Hoff- treated ischemic controls were 2537 ± 980 pg/ml, highermann-La Roche, Inc., Nutley, NJ) 500 ng/kgminute (n than 750 ± 160 pg/ml in sham animals (p <0.05) (Fig.= 9); PGE, (Upjohn Co.) 400 ng/kg/minute (n = 8); the 1); the concentration of 6-keto-PGF, was 317 ± 55 pg/combination of Ibu with the PGI2 analogue (n = 8) and ml, compared with 82 ± 8 pg/ml in sham controls (pIbu with PGE, (n = 8). Sham animals (n = 7) received <0.01) (Fig. 2). After 24 hours, creatinine levels were 4.6saline intravenously. The dosages of the PGI2 analogue ± 0.5 mg/dl, compared with 0.94 ± 0.05 mg/dl in shamand PGE, were the maximum dosages tolerated. Higher animals (p <0.01) (Fig. 3); the weight ofthe ischemic leftdosage resulted in systemic hypotension (data not shown), (L) kidney relative to weight of the previously excisedwhich by itself may lead to renal damage. Neither PGI2 normal right (R) kidney was 118 + 2%, compared with

220 KLAUSNER AND OTHERS Ann. Surg * February 1989

VASODILATING PROSTAGLANDINS

,500

bs

FIG. 2. After 5 minutes of reperfusion, renal ischemia led to high levelsof6-keto-PGF, in saline-placebo-treated ischemic controls. Ibu alone orcombined with the PG12 analogue or PGE, prevented the ischemia-in-duced rise in 6-keto-PGF,. Neither the PG12 analogue nor PGE, aloneaffected 6-keto-PGF, levels relative to placebo-treated ischemic controls.Neither of these agents is degraded to 6-keto-PGF,. Asterisks refer tosignificant differences relative to sham animals, and daggers indicatesignificant differences between placebo- and drug-treated rats.

99 ± 1% in sham animals (p < 0.01) (Fig. 4). Renal his-tology demonstrated severe and extensive changes ofATN(Figs. 5A and B, Table 1).

Pretreatment with Ibu prevented the ischemia-inducedrise in renal TxB2 and 6-keto-PGF, (Figs. 1 and 2). After24 hours, creatinine levels were 4.6 ± 0.4 mg/dl (Fig. 3),the L/R renal weight was 119 ± 5% (Fig. 4), and renalhistology demonstrated extensive ATN (Table 1), changessimilar to those of saline-treated ischemic controls.

Treatment with either the PGI2 analogue or PGE, ledto levels of TxB2, 6-keto-PGF,, creatinine and L/R renalweights similar to those of the placebo-treated ischemiccontrols (Figs. 1-4). Histologic examination in both PG-treated groups revealed extensive changes of ATN (Ta-ble 1).The combination of Ibu and PGE, prevented the rise

in TxB2 and 6-keto-PGF, (Figs. 1 and 2). Compared withischemic controls, creatinine levels were lower (2.3 ± 0.4mg/dl, p < 0.05) (Fig. 3), as was the L/R renal weight(105 ± 3%, p < 0.05) (Fig. 4). Renal histology showedreduced injury (Table 1). The combination of Ibu andPGI2 led to TxB2 and 6-keto-PGF, levels lower than thoseof the sham animals (p < 0.05) (Fig. 3). The L/R renalweight was elevated to 116 ± 5%; however, renal histologyrevealed substantial reduction in the extent ofATN (Figs.SA and B, Table 1).

Discussion

A relatively short, 45-minute period of renal ischemialeads to renal failure, manifest by a four- to fivefold in-

*t *

q ,iFIG. 3. Creatinine levels rose in placebo-treated ischemic controls 24hours after ischemia. Treatment with the PGI2 analogue, PGE,, or Ibualone did not prevent the rise in creatinine, whereas the combination ofIbu with either the PGI2 analogue or PGE, reduced creatinine levels.Asterisks refer to significant differences relative to sham animals, anddaggers indicate significant differences between placebo- and drug-treatedrats.

crease in plasma creatinine levels and microscopic evi-dence of ATN. The 20% gain in renal weight likely rep-resents tissue edema secondary to increased permeabil-ity, an event known to occur in the ischemic reperfusedkidney.9The failure ofthe PG12 analogue or PGE, to attenuate

the renal injury implies that increasing the PG/Tx ratio

130

110

90

- KIDNEY WEIGHT(LT/RT) *

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FIG. 4. Weight of the ischemic left kidney relative to the weight of thepreviously excised normal right kidney rose at 24 hours in placebo-treatedischemic controls Treatment with ibuprofen,.the PGI2 analogue or POE,did not affect the gain in renal weight. Ibuprofen plus PGE1 but not thePG12 analogue modified the increase in renal weight. Asterisks refer tosignificant differences relative to sham animals, and daggers indicatesignificant differences between placebo- and drug-treated rats.

221Vol. 209 * No. 2

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Ann. Surg. February 1989KLAUSNER AND OTHERS

FIGS. 5A and B. Twenty-fourhours after ischemia, kidneysof placebo-treated rats as wellas kidneys of rats treated withIbu alone, the PGI2 analogue,or PGE, showed extensiveATN in the corticomedullaryjunction (A). Kidneys fromanimals treated with Ibucombined with the PG12 an-alogue or PGE, were virtuallynormal (B).

by raising PG concentration without altering Tx is insuf-ficient for reversing the mechanism of ischemic renal in-jury. The correllary observation is consistent with thisthesis, where lowering the PG/Tx ratio by inhibiting Txwithout changing PG is also ineffective in preventingischemia-induced ATN. Lowering the ratio was done by

using the Tx receptor antagonist SQ29,548 (unpublishedobservations).The failure ofvasodilating PGs to protect the reperfused

ischemic kidney may be related to their ability to poten-tiate inflammatory injury and evoke increased vascularpermeability. Thus, vasodilating PGs enhance histamine

222

VASODILATING PROSTAGLANDINS

or complement induced edema in the skin.'10'2 Further,concomitant administration of PGE, with the comple-ment fragment C5a, either by intrabronchial or systemicroutes, results in enhanced pulmonary inflammatory re-actions and increased permeability to protein.'3 Themechanism of the PG potentiation of permeability hasnot been established. Two possibilities appear plausible.First, the adverse effect of PGs may be related to vaso-dilation with exposure ofthe damaged microvascular bedto increased filtration pressure and enhanced edema for-mation.10-14 Secondly, it is possible that the PG-inducedvasodilation and increase in capillary surface area aug-ment the deleterious effects oflocal vasotoxic agents, suchas thromboxanes, by giving them greater access to themicrovascular bed.'0"3 In either case, the potential benefitsof PGs become apparent only when the effects of vaso-toxins are inhibited. This may explain not onlythe inef-fectiveness ofPGI2 analogue or PGE, when administeredalone in renal ischemia, but also their effectiveness whencombined with Ibu, which inhibits TxA2. The failure ofIbu itself to protect the ischemic kidney emphasizes therole of the vasodilating PG. Taken as a whole, these dataindicate that PG and Tx do not act as simple pharma-cologic antagonists, as implied by reliance on the PG/Txratio as a predictor of ischemic injury.The interaction between PGs and TxA2 may involve

an intermediary angiotension (AII).4 The latter is gener-ated in the kidney in response to renal hypoperfusion or

ischemia. 5 The potent vasoconstrictive effects of All are

counteracted in part by synthesis of endogenous vasodi-lating PGs.4" 5 Thus, the increase in renal vascular resis-tance that accompanies an increase in plasma AII levelsis far more pronounced in the presence ofcyclooxygenaseinhibitors such as Ibu.4" 5 Furthermore, the decrease inrenal blood flow induced by cyclooxygenase inhibitorscan be prevented by AII antagonists.'5 It is likely that thelack of protection afforded by Ibu in renal ischemia, de-spite its inhibition ofTxA2 synthesis, is that it blocks PGproduction and allows an unopposed action of All.4 Onthe other hand, interpretation of the effects of pharma-cofogic doses of PGs higher than the maximal estimatesof endogenous secretion rates is complicated by their ca-

pacity to trigger renin release and AII production. Thus,infusion of PGI2 into the renal artery of rats increasesefferent arteriolar resistance, leading to a decline in glo-merular filtration rate.'6 Blocking the renin-angiotensionsystem allows PGI2 to induce a fall in vascular resistancewith increase in glomerular perfusion and filtration. Fur-ther, prostacyclin has been shown to directly stimulaterenin release in man.'7 It is therefore plausible in the cur-

rent study that the direct vasodilatory effects of PGI2 an-

alogue and PGE, were counterbalanced by the vasocon-

strictive effects ofTxA2 and augmented AII synthesis. Thisresulted in a net vasoconstriction, particularly in the vul-

TABLE 1. Acute Tubular Necrosis

Grades

Experimental Group 0 1 2 3 4

Sham ...

Placebo ... ...

Ibuprofen

PGI2-a

PGI2-a + Ibu ... ...

PGE, ....

PGE, + Ibu ... ...

PGI2-a is the analogue ofprostacyclin; each * represents a single kidney.The severity ofATN ranges from a normal of 0 to 4.

nerable region of the kidney, at the corticomedullaryjunction where oxygen supply is tenuous.9"18 Accordingto this thesis, inhibition ofTx synthesis by Ibu along withPG infusion would shift the balance towards vasodilationin the outer medulla and inner cortex, resulting in atten-uation of the ischemic injury.

This postulated mechanism of injury requires that Txlead to regions of selective vasoconstriction. On the otherhand, selective vasodilation must be postulated to accountfor the protective mechanism provided by PGs when givenwith Ibu. Thus, simple vasodilitation induced by non-PGvasodilators such as nitroprusside, even when combinedwith Ibu, does not protect the ischemic kidney.'9 PGs arebelieved to be capable of altering the intrarenal distribu-tion of blood flow. After ischemic injury to the kidney,perfusion is particularly impaired in the corticomedullaryjunction and the outer medulla, where ATN is mostmarked.9"18 The cells of the medullary thick ascendinglimb of Henle, concentrated in this region, are easily in-jured by ischemia because they normally operate at amarginal P02.'8 PGs can redistribute blood flow towardsthese vulnerable regions.9'20

There are two other possible mechanisms of PG pro-tection when TxA2 or All are inhibited. Both TxA2 andPGs can alter the endothelial cell (EC) cytoskeleton. TxA2causes disassembly ofEC actin microfilaments, resultingin cell contraction and widening ofinterendothelial junc-tions, changes associated with increased permeability toprotein and edema formation.2' Depending on their con-centrations, PGI2 and PGE1 do just the reverse, and leadto assembly of actin microfilaments, tightening of inter-endothelial junctions, and promotion ofthe vascular bar-rier, thus preventing ischemia-induced increase in per-meability. The other possible mechanism ofPG protectiontakes place by their anti-aggregatory effects.22 Leukocytes(WBC) are believed to mediate ischemic injury and limit

223Vol. 209 * No. 2

224 KLAUSNER AND OTHERS Ann. Sur -February 1989

blood flow restoration upon reperfusion because of theirrheologic properties and their tendency to adhere to ECduring ischemia.23 Both TxA2 and AII can increase thisWBC adhesiveness.24 PGs are pharmacologic antagonistsand can reduce ischemia induced WBC-EC interactionsand attenuate the injury. This is supported by in vitroobservations using the PGI2 analogue that was found toinhibit WBC adhesiveness and diapedesis through an ECmonolayer.22

These data may have clinical significance. It is alreadyknown that, in humans, cycloxygenase inhibitors do notprotect the underperfused kidney and can actually en-hance injury.4 Given the observation that vasodilating PGsalso augment renin and AII release in humans, indepen-dent ofhemodynamic changes,'7 their recommended6 usein clinical trials ofhypotensive or ischemia-induced renalinjury should be carefully considered.

In summary, the data indicate that ischemia-inducedrenal injury is controlled by a fine balance between TxA2and PGs. Prevention of injury requires manipulation ofboth prostanoids. Thus, administration of vasodilatingPGs attenuate ischemia-induced renal injury only whenTxA2 is inhibited.

Acknowledgment

The authors thanks Ms. Kathleen A. Wilber for her excellent technicalassistance.

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