Role of prostanoids and endothelins in the prevention of cyclosporine-induced nephrotoxicity

Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(4&5), 231^239 & 2001 Harcourt Publishers Ltd doi:10.1054/plef.2001.0265, available online at http://www.idealibrary.com on

Role of prostanoids and endothelins in the prevention of cyclosporineinduced nephrotoxicity I. E. Darlametsos, D. D.Varonos Centre Franco-Helle¤nique de Recherches Biome¤dicales hhNikolaos Papanikolaouii, Corporation of the Municipality Agrinion, 30100 Agrinion, Greece

Summary Cyclosporine A nephrotoxicity includes both functional toxicity and histological changes, whose seriousness is dependent upon the dose and the duration of the drug administration. Several vasoactive agents have been found to be implicated in cyclosporine induced nephrotoxicity, among which prostanoids and endothelins are the most important.In previous studies we were able to prevent the early stage (7 days) of cyclosporine (37.4 mmol [45 mg]/kg/day) induced nephrotoxicity in rats either by the administration, i) of OKY-046, a thromboxane A2 synthase inhibitor, ii) of ketanserine, an antagonist of S2 serotonergic, a1 adrenergic, and H1 histaminergic receptors and iii) of nifedipine, a calcium channel blocker, or by diet supplementation either with evening primrose oil or fish oil. All these protective agents elevated ratios of excreted renal prostanoid vasodilators (prostaglandins E2, 6ketoF1a) to vasoconstrictor (thromboxane B2), a ratio which was decreased by the administration of cyclosporine alone. Nifedipine averted the cyclosporine induced increase of urinary endothelin-1release. All protections were associated with the reinstatement of glomerular filtration rate forwards normal levels whereas renal damage defence, consisting ofa decrease ofthe cyclosporine inducedvacuolizations, wasvariable.Ketanserine and evening primrose oilwere the onlyagents which prevented the animal body weight loss.These data suggest that prostanoids and endothelin-1may mediate functional toxicity while thromboxane A2 is involved the morphological changes too, provoked in the early stage of cyclosporine treatment. However, other nephrotoxic factors and additional mechanisms could also be implicated in the cyclosporine induced nephrotoxicity. & 2001Harcourt Publishers Ltd

CYCLOSPORINE NEPHROTOXICITY Cyclosporine A (CsA) is a widely used drug in the prevention of allograft rejection as well as in the treatment of some autoimmune diseases.1,2 In contrast to other available immunosuppressants, CsA reversibly inhibits only some classes of lymphocytes and does not affect haemopoietic tissues.2 However, the use of CsA in immunosuppressive therapy has proven to be a double edged sword on account of its diverse organ side-effects, among which nephrotoxicity (NT) and hypertension are

Received 3 January 2001 Accepted 17 January 2001 Correspondence to: I. E. Darlametsos, Centre Franco-Helle¤nique de Recherches Biome¤dicales PO Box 57, L. Mavili 6, 30100 Agrinion, Greece. Tel.: 0641-45 760; Fax: 0641-39 398; E-mail: [email protected]

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the most common and grave.3–6 Cyclosporine NT is so serious that limitation of the drug use has been proposed.4 At any time during CsA treatment renal dysfunction may occur, characterised by a spectrum of renal injuries classifiable into functional toxicity and morphological changes. CsA-induced functional toxicity consists of a dose-dependent reduction of renal plasma flow (RPF) and glomerular filtration rate (GFR), which are reversible upon drug dose diminution.7–9 It is well known that CsA provokes intense vasoconstriction in the kidney. However, the mechanism of its action remains under debate. Numerous vasoactive agents have been incriminated since CsA nephrotoxicity (NT) was found to be associated with the enhanced production of many vasoconstrictor factors such as renin–angiotensin (R–A),10 noradrenaline (NA),11 thromboxane A2 (TXA2)12,13 endothelin (ET)14,15 and the reduced release of vasodilator prostaglandins E2 and I2 (PGE2 and PGI2,

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known as prostacyclin).13–16 However, contrasting experimental data display CsA nephrotoxicity partially independent of changes in prostanoid (PGE2, 6ketoPGF1a and TXB2 which are the stable metabolites of PGI2 and TXA2, respectively) syntheses, potentially due to variation of rat strains.17,18 Concerning renal morphological changes, CsA reveals a damage spectrum of varied severity. The tubular changes, consisting of vacuolization, megamitochondria and microcalcification, are reversible. Arteriolar changes may progress to focal interstitial fibrosis and nephron loss.19 However, it remains unclear whether irreversible changes are dependent on haemodynamic modifications, tubular damage or both. Despite numerous studies, the elucidation of the mechanism by which CsA induces clinical nephrotoxicity persists in being an important issue for further investigation. Subsequently, we refer to the contribution of the more important constrictor factors implicated in the CsA induced nephrotoxicity.

as much by adrenergic tone pharmacological blockade as by renal denervation.30 Large doses of NA injection, in the renal artery, induced ARF.29,31 Nevertheless, it appears unlikely that excessive local release of catecholamines is the primary cause of haemodynamic deviations of ARF, since H. Eliahou et al.32 showed that ARF could be induced in totally denervated, transplanted kidney, and was not prevented or reversed by adrenergic blockade. Furthermore, N. Papanikolaou et al.33 observed that depletion of endogenous catecholamines with reserpine did not protect rats against ARF induced by glycerole. With regard to the induction of ARF by the enhanced release of vasoconstrictor substances, increasing preglomerular resistance such as renin–angiotensin (R–A) and noradrenaline (NA), either (R–A) play a secondary role in the development of renal dysfunction or its (NA) involvement is under debate. By contrast, other vaso active substances like prostanoids and endothelins (see below) seem to play pivotal roles in ARF development.

ROLES OF RENIN–ANGIOTENSIN SYSTEM AND NORADRENALIN

PROSTANOIDS

It is widely believed that afferent arteriole contraction rather than renal tubular damage is the major pathogenetic factor in CsA induced nephrotoxicity (NT).20 This concept is compatible with the fact that the drug NT largely reflects vascular phenomena21 dictated by vasoactive agents. Plasma renin activity (PRA) was found increased22, 23 or decreased24 in animals after CsA administration while CsA ingestion by healthy human volunteers provoked a GFR fall, which correlated significantly with PRA suppression.25 But, the renin–angiotensin (R–A) system antagonism ameliorated renal haemodynamic harmed by CsA in rats.26 During CsA (15 mg/kg/day) administration in rats, angiotensin converting enzyme (ACE) inhibition exacerbated the glomerular dysfunction even beyond that caused by CsA alone, from the third till the fifth week of CsA treatment, while it ameliorated the tubular interstitial fibrosis (but not dilatation or vacuolization) at the fifth week.27 Such observations rather suggest a role for angiotensin II in renal morphological changes. However, in the case of glycerol, another agent inducing acute renal failure (ARF), N. Papanikolaou et al.28 have shown that the ACE inhibition did not protect the rats against ARF while the selective inhibition of TXB2 synthesis did protect them. Furthermore, it is consistent with this observation that injection of large doses of angiotensin II into the renal artery did not provoke ARF.29 On the other hand, CsA was found to augment the production of noradrenaline (NA)11. Now, the increase of renal vascular resistance, induced by CsA, was prevented

The prostanoids, prostaglandins (PGs) and thromboxanes (TXs), are cyclic eicosanoids. They are derived from both types of essential fatty acids (EFAs) the o-6 and the o-3 originated from linoleic acid (LA) and alpha-linolenic acid (ALA), respectively. Notably LA is desaturated to gammalinolenic acid (GLA), rapidly elongated to dihomogammalinolenic acid (DGLA), which is in turn slowly desaturated to arachidonic acid (AA) while ALA with alternating desaturation and elongation steps gives eicosapentaenoic acid (EPA).34 DGLA and AA are the main o-6 EFAs eicosanoid precursors while EPA is the main o-3 EFA eicosanoid precursor. The bulk of these EFAs in mammalian cells (mainly AA) is esterified in the membrane cell phospholipids, where it is stored until its release by the activation of phospholipases C and A2 (PLC and PLA2).35 The EFAs availability from membrane phospholipids may control PG syntheses. So, AA through the cycloxygenase pathway is converted to the endoperoxides PGG2 and PGH2, which gives rise to the 2 series of PGs (PGE2, PGF2a, PGD2, PGI2 and TXB2) while through the lipoxygenases12,5,15 or 11 gives rise to Leukotrienes (LTs) and to hydroxy-or hydroperoxy-eicosatetraenoic acids (HETEs or HPETEs, respectively).36 When DGLA or EPA are the substrata then through the cycloxygenase pathway the 1 (PGE1, TXA1 etc) or 3 (PGE3, TXA3 etc) series, respectively, of PGs are synthesized. The prostanoids are shortlived and biologically active molecules. Most PGs are vasodilators with favourable effects in human and other species peripheral vascular beds. Their role is important in the maintenance of renal function. Reduced PG-synthesis is accompanied by a decrease of GFR and sodium excretion rate.37

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PG-synthesis in the medulla is higher than in the cortex and its decrease occuring in kidney disease depends on the location, specificity and stage of the disorder.37, 38 In the acute phase of some illnesses a stimulation of PGsynthesis has been observed while during the later stages its decrease was found.37 Infusions of PGE1, PGE2 and PGI2 protected against ARF39–42 while PGE1 infusion in patients with chronic renal disease improved their renal function.43 Furthermore PGE1 almost completely prevented HgCl2-induced glomerulonephritis.44 Thromboxane A2 is another prostanoid, whose dominant biological properties are opposite those of prostacyclin i.e. it is a potent vasoconstrictor and platelet aggregator agonist. These properties are shared by neither TXB1 nor TXB3, which are inactive.37 TXA2 is predominantly synthesized by circulating platelets.45 Within the glomerulus, it may also be synthesized by messangial and epithelial cells.46,47 The pathogenetic role of this prostanoid is well known in some types of ARF, where it acts as a mediator of kidney vasoconstriction. TXA2 is the major cause of renal vaso-constriction induced by different chemical compounds such as glycerol, HgCl2 and aminoglycosides.48,49,50 ROLE OF PROSTAGLANDINS AND THROMBOXANES Concerning CsA administration, reduced production was observed either of PGI2 by endothelial cells or of cyto-

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protective PGE2 in glomeruli and renal medulla.51,52,53 These findings are consistent with our previous observations of the diminished urinary PGE2 and 6 kPGF1a (6 ketoPGF1a) production (Table 2) after the short term (7 days) CsA (37.4 mmol [45 mg]/kg/day) treatment of rats.13 Additionally, we observed enhanced excretion of urinary TXB213 as did other investigators.54 In these studies the kidneys of all rats which received CsA displayed functional toxicity as well as morphological changes, being confirmed by a fall in GFR (Table 1) or by tubular vacuolizations (mainly) (Table 3), respectively. We could prevent this early stage of CsA induced nephrotoxicity by rat diet supplementation with i) evening primrose oil (EPO)55, an EFA containing GLA, that is a precursor of PGE1 synthesis or ii) fish oil (FO),55 an EFA containing EPA, that is the precursor of PGE3 production and also by the administration of drugs i) OKY-046,56 a TXA2-synthase inhibitor, ii) of ketanserine (KTS),13 an antagonist of S2 serotonergic (mainly), a1 adrenergic and H1 histarninergic receptors and iii) of nifedipine (NFD),56 a dihydropyridine-sensitive Ca2þ channel blocker, in animals. The protection offered to the animal kidneys, by the use of EPO, FO, OKY-046 and KTS, was ascertained both by the restoration of GFR to normal levels (Table 1) and by the prevention of renal lesions, mainly diffuse vacuolization (Table 3), caused by CsA alone.13,55,56 These efforts to prevent the acute phase of CsA induced

Table 1 Creatinine clearance (Ccr), body weight change (BWC) and urinary prostanoid ratios of normal rats (NR) and cyclosporine A (CsA) or CsA þ a renoprotective mean treated animals Animal group

Ccr ml/Kg/min

BWC %

6kPGF1a/TXB2

PGE2/TXB2

NR CsA CsAþEPO CsAþFO CsAþOKY CsAþKTS CsAþNFD

2.53+0.14*** 1.19+0.10 2.69+0.13*** 2.18+0.13*** 2.47+0.11*** 2.56+0.28*** 2.63+0.31***

þ0.63+0.44*** 712.50+1.96 76.76+1.43* 710.37+1.58 79.92+1.61 77.15+1.71* 711.16+2.64

1.52+0.26*** 0.42+0.05 2.36+0.50** 1.55+0.33** 4.84+1.67** 0.76+0.17* 0.90+0.21*

4.13+0.71*** 0.67+0.09 3.73+0.54*** 3.61+0.8 1** 6.32+1.20*** 1.40+0.27* 1.72+0.41*

6 keto prostaglandin F1a (60kPGF1a ), thromboxane B2 (TXB2), prostaglandin E2 (PGE2) are prostanoids. Evening primrose oil (EPO), fish oil (FO), OKY-046 (OKY), ketanserine (KTS) and nifedipine (NFD) are renoprotective means.Values are means + SEM; n = 9. All values were compared against those obtained in the second (CsA) group. Significantly different from CsA group values at: *P50.05, **P50.01and ***P50.001. Table 2 Urinary prostanoid and endothelin levels of normal rats (NR) and cyclosporine A (CsA), or CsA þ a renoprotective mean treated animals Animal group

6kPGF1a pmol/kg/24 h

PGE2 pmol/kg/24 h

TXB2 pmol/kg/24 h

ET-1 fmol/kg/24 h

NR CsA CsAþEPO CsAþFO CsAþOKY CsAþKTS CsAþNFD

406+30* 314+30 738+124** 624+85** 974+200** 469+128 413+47*

1087+70*** 512+70 1307+191** 1342+188*** 1448+225*** 770+130 793+81**

297+27*** 806+86 396+68** 453+57** 317+57*** 598+59* 650+162

121+13** 468+120 ^ ^ ^ ^ 206+16*

6 keto prostaglandin F1a (6kPGF1a), prostaglandin E2 (PGE2), thromboxane B2 (TXB2), endothelin-1 (ET-1).Evening primrose oil (EPO), fish oil (FO), OKY-046 (OKY), ketanserine (KTS) and nifedipine (NFD) are the renoprotective means.Values are means + SEM;.n = 9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.05, **P50.01and ***P50.001.

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Table 3 Parameters appreciated in light microscopic sections of normal rats (NR) and cyclosporine A (CsA) or CsAþ a renoprotective mean treated animals Animal group

AR

NR

1 2 3 1 2 3 4 5 6 7 8 9 1 2 3 4 5 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7

CsA

CsA þ EPO

CsA þ FO

CsAþ OKY-046

CsA þ KTS

CsA þ NFD

Dif-VCL-Foc + þ 7 7 2þ 3þ 3þ 7 2þ 2þ 7 + 7 2þ 7 7 2þ þ 2þ 7 7 þ þ 2þ 7 7 7 3þ 2þ + 2þ þ þ 7 3þ þ + 7 3þ 2þ 7

7 7 7 þ 7 7 7 + 7 7 2þ 7 þ 7 2þ þ 7 7 7 þ 7 7 7 7 þ þ 2þ 7 7 7 7 7 7 þ 7 7 7 2þ 7 7 4þ

BBL

SCN

TBCT

ITIF

ITOED

DLT

7 7 7 7 2þ þ 2þFoc 7 7 7 þ 7 7 7 + 7 7 7 7 7 þ 7 7 7 7 7 7 7 7 7 + 7 7 7 þFoc 7 7 þ2Foc 7 7 7

7 7 7 7 2þ + + 7 7 7 + 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 + 7 7 7

7 7 7 7 þ 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

7 7 2þ 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

+ 7 7 7 7 þ þ 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 þ 7 7 þ 7 7 7

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 þ 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Affected rats (AR), tubulardiffuse -vacuolization- or focal (Dif-VCL-Foc), brush border loss (BBL), single cellnecrosis (SNC), tubular casts (TBCT), interstitialinfiltration (ITIF), interstitialoedema (ITOED), dilatation (DLT).Crossesindicate the score on a 0^4 scale.Evening primrose oil (EPO), fish oil (FO), OKY-046 (OKY), ketanserine (KTS) and nifedipine (NFD) are the renoprotective means.

nephrotoxicity (NT) have shown the strong prostanoid implications since protection was always accompanied by a reduction of the vasoconstrictor TXB2 (TXA2) excretion as well as the enhanced production of renal vasodilators PGE2 (and/or PGE1 for EPO, and/or PGE3 for EPA) and 6 kPGF1a,, (PGI2) (Table 2).55, 56 However, in the case of KTS the observed PGE2 and 6 kPGF1a rises were not statistically significant while the prostanoid ratios of vasodilators (PGE2 and 6 kPGF1a) to vasoconstrictor (TXB2) were (Tables 1 & 2).13 The EPO protection may be due to GLA, which augments the DGLA levels, without affecting the AA ones. The DGLA increase gives rise to PGE1 (potent vasodilator, antiinflammatory, antiaggregatory agent while TXA1 is inactive) synthesis, and redirects PGH2

conversion to its protective prostaglandin E2, I2 metabolites instead of TXA2 (powerful pro-aggregatory vasoconstricting substance).34, 55 The mechanism of FO protection must be due to the EPA, which is contained in FO. EPA is the precursor of the three series of prostanoids, notably the vasodilator and anti-aggregatory PGE3 and the inactive TXA3. Thus, the ways by which EPO and FO induce rises of PGE1,2 and PGE2,3 are becoming clearer. The kidney’s defence via thromboxane synthase inhibition, may be due to the redirection of AA metabolism, through PGH2, to vasodilator PGE2, PGI256, 50 in lieu of TXA2. EPO and KTS were the only agents which prevented the body weight loss (Table 1) of the animals.56 The better defensive effects obtained by EPO may be due to PGE1

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release because, its beneficial actions, including increase of renal haemodynamics or inhibition of TXA2 and noradrenaline release as well as platelet aggregation, seem to establish PGE1 as the most protective prostaglandin against the development of hypertension and renal disorders.37 KTS is an antagonist of vasoconstrictor S2 serotonergic receptors, but not of vasodilator S2 ones, and appears to be a new therapeutic tool for investigating the role of serotonin (5-HT) in the pathogenesis of ARF.13 Additionally KTS antagonizes the amplifying effects of 5-HT on other vasoconstrictor substances such as catecholamines and histamine (by antagonizing a1 adrenergic and H2 histaminergic receptors, respectively) and PGF2a.57 Thus, KTS major protection against CsA-induced NT could be attributed not only to TXB2 reduction (Table 2) but also to KTS’s defensive actions against both serotonin and serotonin’s amplifying effects on other vasoconstrictor substances (such as noradrenaline and PGF2a). With regard to the partial protection brought about by NFD, this may be due to augmented ratios of urinary prostanoid vasodilators (PGE2 and 6kPGF1a) to vasoconstrictors (TXB2) as well as to reduced release of renal origin ET-1 (Tables 1 & 2).15 NFD only slightly prevented the CsA induced increase of TXB2 levels. This could explain why this protection relates mainly to functional toxicity and hardly to structural renal damage, mediated at least in part by the preservation of high renal TXB2 levels. TXA2, in addition to its potent renal vasoconstriction, favours platelet aggregation, also implicated in the glomerular injury.58–61 Consequently, TXA2 may mediate both functional toxicity and structural renal damage induced by short term CsA treatment. Other investigators have observed that the administration of PGE2 analogues or TXA2 synthesis inhibition improved the manifestations of experimental CsA nephrotoxicity.62, 63 The suppression of TXA2 production was found protective in humans too, since studies in FOtreated renal transplant recipients, undergoing immunosuppressive therapy, showed both a reduction of rejection episodes and a better recovery of kidney function.64,65 The renal function improvement in CsA-treated allograft patients receiving a thromboxane synthase inhibitor, confirms the relevance of TXA2 to pathogenesis of this syndrome.66 ROLE OF ENDOTHELINS ETs form a 21 aminoacid peptide family containing four isoforms, ET-1, ET-2, ET-3 and the vasoactive intestinal contractor (VIC), and are produced in a broad variety of tissues of all species.67 These peptides reveal a wide diversity of biological actions, via binding to their receptors.67 Actions include potent vasoconstriction of & 2001Harcourt Publishers Ltd

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several vascular beds, specifically of the renal one,68 mitogenesis, cell proliferation and hormone production. ET-1, the only family member produced in endothelial cells,69 is the most potent vasoconstrictor peptide known to date.70 Tubular,71 mesangial72 and several renal epithelial cells73 additionally to endothelial ones produce ET-1. Numerous studies showed that ET-1 is implicated in the regulation of renal physiology as well as in the pathogenesis of CsA-, HgCl2- and glycerol-induced ARF21,74–76. CsA enhances urinary ET-115,77 and also renal ET receptor number77 in rats. Furthermore, it has been proposed that ET can contract afferent and efferent arterioles78,79 resulting in NT. Therefore, the CsA induced NT is probably caused in part by the increased production of ET-115,21,80. Anti-ET serum infusion21 prevented glomerular vasoconstriction following CsA administration. Moreover, endothelin receptor antagonism was protective in CsA induced NT27, 81. Kon et al.27 noted that the combined antagonism of endothelin A/B receptors prevented only renal functional toxicity but not structural damage, associated with the long term treatment with CsA. This shows that ET action is linked to vasoconstriction, which is compatible with our observation15 that nifedipine administration in rats subjected to short term CsA treatment, decreased the urinary and renal origin ET-1 and restored the GFR (Tables 1 & 2). But, it only marginally prevented the histological renal changes induced by CsA (Table 3), possibly due to the preserved high levels of renal TXA2 (TXB2). IS ENDOTHELIN VASOCONSTRICTION CYCLOXYGENASE DEPENDENT DURING CYCLOSPORINE TREATMENT? ET-1 has been shown to promote the release of various vasoactive substances including endothelium relaxing factor (nitric oxide),82, 83 atrial natriuretic peptide and arginine vasopressin84,85,86 as well as such prostanoids as 6 kPGF1a, PGE2 and TXA2.87,88 ET-1 binding to its receptor sites possibly activates phospholipases PLA2 and PLC, resulting in prostanoid production.89 Potential interactions of the locally generated autacoids with ET could moderate or aggravate the peptide’s vasoconstricting actions. Of interest seems to be the role of cycloxygenase products in endothelin-induced responses of the kidney since ET has been shown to augment renal prostacyclin, PGE2 and PGF2a.79, 89, 90 In vitro studies indicated that the use of OKY-046 had no effect on the isolated, afferent and efferent, arteriole constriction induced by exogenously administered ET-1.79 So, in our studies even if the augmented renal ET-1 release, caused by the CsA administration, persists during OKY-046 administration then the ET-1 induced

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renal contraction could not be mediated by TXA2, whose local generation falls (Table 2).56 Other observations have shown that selective TXA2 antagonism did not prevent the diminution of GFR during ET infusion in rats while cycloxygenase inhibition did. These data indicate that prostanoids other than TXA2, possibly PGE2a, play a key role in sustaining ET induced renal vasoconstriction. Furthermore, the observed release of vasodilator PGE2, was of minor importance.79 So, ET may produce its renal vasoconstrictor effects either per se or by synergism with other factors such as PGF2a, which also exert contractile actions in the rat kidney.79 However, the combined administration of CsA with indomethacin provoked stronger renal dysfunction in humans than did CsA alone.91 We found that the CsA administration in rats increased renal ET-1 and TXA2 production. But the peptide’s rise did not enhance PGI2 and PGE2 release (Table 2). This indicates that the prostanoid releases due to PLC and PLA2 activation induced by the peptide’s binding to its receptors may be of secondary import. Analogous effects were revealed again in our observations that NFD administration in rats receiving CsA provoked only a reduction in ET-1, which was not accompanied by a fall either in TXA2 or in PGI2 and PGE2 (Table 2).15,56 These data may suggest the existence of independent endothelin and prostanoid routes of vasoactive actions or the existence only of secondary interactions between them in CsA-induced NT. SUMMARY AND CONCLUSIONS The more important vasoactive factors, known to date, contributing to cyclosporine NT are the cycloxygenase metabolites and endothelins. Rise of both powerful renal vasoconstrictors TXB2 (TXA2) and ET-1 as well as reductions of renal vasodilators PGE2 and 6kPGF1a (PGI2) have been observed during CsA treatment. The partial protection against the acute phase of CsA NT, by the use of EPO, FO, OKY-046 and KTS, producing a reduction in renal TXA2 synthesis, was obtained in rats. All the means of protection, with the exception of KTS, were associated with a rise of renal PGE2 (PGE1,2 for EPO and PGE2,3 for FO) and PGI2 syntheses. However, KTS like the other protective agents, did provoke a rise in the ratios of prostanoid vasodilators (PGE2, PGI2) to vasoconstrictors (TXA2). There are no data about the levels of the released ET-1. These protective agents concern both functional toxicity and structural damage induced by short term CsA treatment. KTS and EPO were the only means which prevented additionally the body weight loss of CsA treated animals. Potentially ketanserine’s efficiency could be due to its more extensive protective action on serotonin, or to serotonin’s amplifying effects

on other constrictor factors (such as PGF2a and noradrenaline). The effectiveness of EPO may be due to the major beneficial properties of PGE1, which seems to be the most protective prostaglandin in kidney diseases. The NFD protection offered in rats receiving CsA was accompanied by a fall of the ET-1 release. This protection was largely functional since the CsA-induced renal lesions were only slightly prevented. This may be related to the preserved high TXB2 levels despite the observed rise of the ratios of renal vasodilators (PGE2, 6kPGF1a) to vasoconstrictor TXB2. Given that only secondary interactions at present appear to exist between ET-1 and cycloxygenase products in relation to CsA NT, joint studies on both thromboxane and endothelin systems are required for the elucidation of their contributions to the NT induced by long term CsA treatment. It remains uncertain whether the combined regulation of the potent vasoconstrictor components (TXA2 and ET-1) of both systems might have more beneficial effects on immunosuppressive therapy than either alone. However, other nephrotoxic factors, known or unknown, and additional mechanisms could also be implicated in CsA-induced NT.

ACKNOWLEDGEMENTS The authors are indebted to Dr P. Morphake for light microscopy examinations. We thank also Mrs E. Papanikolaou and Mrs D. Markopoulou for their secretarial assistance, Mrs E. Tsakanika for her administrative assistance and Mr P. Papadogeorgopoulos for his help with English.

REFERENCES 1. Moller E. Areas for further experimentation to elucidate the immunosuppressive activity of cyclosporine. Transplantation 1988; 46: 20S–23S. 2. Rehacek Z., Zhao D. The biochemistry of cyclosporin formation: a review. Proc Biochem 1991; 26: 157–166. 3. Klintmalm G. B. G., Iwatsuki S., Starzl T. E. Nephrotoxicity of cyclosporine A in liver and kidney transplant patients. Lancet 1981; i: 470–471. 4. Myers B. D. Cyclosporine nephrotoxicity. Kidney Int 1986; 30: 964–974. 5. Curtis J. J., Luke R. G., Jones P., Diethelm A. G. Hypertension in cyclosporine-treated renal transplant recipients is sodium dependent. Am J Med 1988; 85: 134–138. 6. First M. R., Neylan J. F., Rocher I. L., Tejani A. Hypertension after renal transplantation. J Am Soc Nephrol 1994; 4(suppl 8): S30–S36. 7. Sabatini M., De Nicola, Sansone G., Conte G. Renal hypoperfusion as the primary cause of cyclosporine-induced nephropathy. Nephrol Dial Transplant 1993; 8: 794–797. 8. Paller M. S., Ferris T. F. Effects of Nva2-cyclosporine on glomerular filtration rate and renal blood flow in the rat. Transplantation 1987; 43: 893–895.

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