The role of phospholipase A2 and cyclooxygenase in renal toxicity
A.C.L. Nobrea, G.R. Coeˆlhoa, M.C.M. Coutinhoa, M.M.M. Silvaa, E.V. Angelima,
D.B. Menezesb, M.C. Fontelesc, H.S.A. Monteiroa,*
aDepartment of Physiology and Pharmacology, Federal University of Ceara, Rua Cel. Nunes de Melo, 1127, 60430-270 Fortaleza, CE, Brazil
bDepartment of Pathology and Forensic Medicine, Federal University of Ceara, Rua Monsenhor Furtado, S/N,
cCeara State University, Av. Paranjana, 1700, Campus do Itaperi, 60740-000 Fortaleza, CE, Brazil
Received 14 February 2000; accepted 12 June 2000
We have shown previously that exposure to microcystin-LR (MCLR) causes renal toxic effects in isolated perfused rat
kidney. That study was extended further to approach the perspective of pharmacological blockade of renal toxic effects byMCLR through the use of experimental therapeutic agents. An isolated kidney perfusion system was utilized and samples ofurine and perfusate were collected at 10 min intervals to determine the levels of inulin, sodium, potassium and osmolality. Dexamethasone (20 mg mlϪ1) and indomethacin (10 mg mlϪ1) were administered in the beginning of the perfusion and MCLRwas employed in a dose of 1 mg mlϪ1 after an internal control of 30 min to evaluate the perfusion pressure (PP), renal vascularresistance (RVR), glomerular filtration rate (GFR) and urinary flow (UF). Dexamethasone and indomethacin antagonized thetoxic effects of MCLR on PP, RVR, GFR and UF. Histologic analysis of dexamethasone and indomethacin treated groups didnot show any vascular or interstitial alterations. MCLR potentially impairs the renal function, probably causing vascular andglomerular lesions and, promoting renal alterations through direct or indirect actions. These data seem to indicate that the renalalterations promoted by MCLR involves also phospholipase A2 and arachidonic acid-derived mediators. ᭧ 2000 ElsevierScience Ltd. All rights reserved. Keywords: Microcistin-LR; Perfused kidney; Dexamethasone; Indomethacin
There are few papers in the literature that show the effect
the proximal tubular epithelium with slight tubular dilata-
of microcystin-LR (MCLR) in the renal system. Radbergh et
tion (Hooser et al., 1989) and cellular damage were also
al. (1991) have shown degenerative changes in the tubular
found in glomeruli and tubules (Radbergh et al., 1991).
epithelial cells, glomeruli and interstitial tissue in kidneys of
The utilization of the perfusion system of isolated kidney
carps exposed to MCLR, via intraperitoneal. In contrast to
permits the study of the direct effect of toxin without
what occurs in fish, which presents more severe changes,
systemic interference. Our laboratory showed that MCLR
renal damages are seldom observed in mice and this can be
is capable of changing the kidney functional parameters
explained by the survival time to lethal dose. Kotak et al.
(perfusion pressure (PP), renal vascular resistance (RVR),
(1996) showed renal lesions in fishes (Oncorhynchus
glomerular filtration rate (GFR) and urinary flow (UF)) in
mykiss) that consisted of coagulative tubular necrosis and
isolated perfused rat kidney (Nobre et al., 1999). The scope
dilatation of Bowman’s space. Glomerular capillaries were
of this work was to approach the perspective of pharmaco-
filled with eosinophilic fibrillary material after nine hours of
logical blockade through the use of experimental therapeutic
exposure to MCLR. There has been a mild vacuolization of
MCLR (Sigma Chemical Co., Saint Louis, MO, USA;
Molecular mass 995.2) was used in the current experiments
* Corresponding author. Fax: ϩ55-85-2815212. E-mail address: [email protected] (H.S.A. Monteiro).
obtained from Microcystis aeruginosa. Adult Wistar rats of
0041-0101/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 1 9 3 - 8
A.C.L. Nobre et al. / Toxicon 39 (2001) 721–724
Table 1Effects of dexamethasone and indomethacin in the renal toxicity caused by MCLR in perfused rat kidney (Results are expressed as means ^SEM of Control kidneys (n 7) versus MCLR treated kidneys (n 7) and treated as: MCLR microcystin-LR (1 mg mlϪ1); Dexa dexamethasona (20 mg mlϪ1); Indo indomethacin (10 mg mlϪ1). PP perfusion pressure; RVR renal vascular resistance; UF urinaryflow; GFR glomerular filtration rate. The first 30 min represent the internal control for each group)
a Significance in relation to control (p Ͻ 0,05).
b Significance in relation to treated group with MCLR (p Ͻ 0,05), compared by ANOVA (Bonferroni test).
both sexes weighing 250–280 g were anesthetized with
pentobarbital sodium (50 mg kgϪ1 body weight ip). Before
(10 mg mlϪ1) studies were initiated before an internal
the experiment, the animals were fasted for 24 h with access
control of 30 min, and MCLR (1 mg mlϪ1) after an internal
to water ad libitum. The right renal artery was cannulated as
control, and observations were made during the next 90 min.
described by Bahlmann et al. (1967), Nishiitsutji-Uwo et al.
Naϩ and Kϩ were determined by flame photometry (Flame
(1967) and Ross (1978). The perfusate was a modified
Photometer IL, Model 445), and inulin was analyzed
Krebs–Henseleit solution with the following composition
according to Walson et al. (1955) as modified by Fonteles
in mmol lϪ1: Naϩ 147, Kϩ 5, Caϩϩ 2.5, Mgϩϩ 2, ClϪ 110,
et al. (1998). Osmolality of the samples was measured in an
Advanced Instruments osmometer (Needham Heights, MA).
urea and 0.075 g inulin. Six grams of bovine serum albumin
After the renal perfusion a histologic evaluation of kidneys
(BSA fraction V, Sigma) were added to the solution after a
was made by optical microscopy. Dexamethasone was
previous dialysis for 48 h at 4ЊC in 1.5 l of Krebs. The pH
acquired from Merck and CO., Inc (EUA) and indomethacin
was then adjusted to 7.4. Total perfusate used per experi-
The data were presented as mean ^ S.E.M. At least seven
The perfused rat kidney model followed the method of
different animals were used for each data point. Data were
Bowman (1970) as modified by Fonteles et al. (1983, 1998).
analyzed by ANOVA (Bonferroni test). For statistical
The system was calibrated for flow and resistance before
purposes, p Ͻ 0:05 was considered significant.
each experiment. The rate of perfusion flow was maintained
Previous results showed that infusion of 1 mg mlϪ1 of
of 25–35 ml minϪ1 per kidney. The first 30 min of perfusion
MCLR after 30 min of internal control caused alterations
were considered to be an internal control. Each experiment
of renal functional parameters (Nobre et al., 1999).
was divided into four periods of 30 min; these periods were
Dexamethasone (20 mg mlϪ1) showed a capacity of
further subdivided into equal intervals of 10 min. During
reverting the renal changes promoted by MCLR when
each 10 min period, samples of perfusate and urine were
compared to the control group and internal control
collected for determinations of sodium, potassium, inulin
(Table 1). The drug was able to antagonize in a significant
manner p Ͻ 0:05 the effect on the PP, RVR, UF and GFR
A.C.L. Nobre et al. / Toxicon 39 (2001) 721–724
at 60, 90 and 120 min. Indomethacin at the dose of
1998). In our experiments we have observed that MCLR
10 mg mlϪ1 was able to protect the kidneys from the toxic
seems to induce the activation of phospholipase A2 and
effects of MCLR as compared with the control group and the
cyclooxygenase as its effects were blocked by dexamethasone
internal control group (Table 1). A significant reversion of
and indomethacin; this mechanism is similar to the hepatic
the effects of MCLR on the PP, RVR, UF and GFR at 60, 90
effect. It is supposed that this renal change occurs probably
by damaging both vascular and glomerular sites.
Nobre et al. (1999) showed an intense amount of protei-
naceous material in the urinary spaces following perfusionwith MCLR. Proteinaceous material was not seen in the
tubules or urinary space of kidneys pretreated with dexa-methasone and indomethacin, nor were there any abnorm-
Acknowledgments are made to Maria Sı´lvia Helena
alities in the renal vessels and interstitium, suggesting a
Freire Franc¸a and Domingos Barreto Oliveira for technical
protective effect of these substances.
assistance. This research was supported by CNPq and
Naseem et al. (1990) evaluated the effects of glucocorti-
coids in the release of arachidonic acid and its metabolitesinduced by MCLR in rat hepatocytes. They showed thatfluorcinolone, dexamethasone and hydrocortisone suppress
the release of arachidonic acid, prostacyclin (6-ketoF1a) andTXB
Bahlmann, J., Giebish, G., Ochwadt, B., Schoeppe, W., 1967.
2. It is known that microcystins are capable of inducing
the cyclooxygenase via the metabolism of arachidonic acid
Micropuncture study of isolated perfused rat kidney. Am. J. Physiol. 212, 77–82.
thus being prone undergoing the action of glucocorticoids.
Bowman, R.H., 1970. Gluconeogenesis in the isolated perfused rat
In our experiments, MCLR induces an increase in PP
kidney. J. Biol. Chem. 245, 1604–1612.
which was inhibited by indomethacin. The results suggest
Braquet, P., 1987. Perspectives in platelet activation factor research.
that this increase in pressure can be related to alterations in
the homeostasis of cell calcium. If MCLR induces change in
Fonteles, M.C., Cohen, J.J., Black, A.J., Wertheim, S.J., 1983.
the calcium metabolism of smooth muscle cell, this change
Support of renal kidney function by long-chain fatty acids
in calcium input can be contributing to an arteriolar vaso-
derived from renal tissue. Am. J. Physiol 244, 235–246.
Fonteles, M.C., Greenberg, R.N., Monteiro, H.S.A., Currie, M.G.,
Calcium plays an important role in the activation of phos-
Forte, L.R., 1998. Natriuretic and Kaliuretic activities of guany-
lin and uroguanylin in the isolated perfused rat kidney. Am. J.
2. If MCLR influences the activity of calcium, it
Physiol. 275, F191–F197 (Renal Physiol 44).
is possible that it also intensifies the activation of this
Haystead, C.M., Gailly, P., Somlyo, A.V., Haystead, T.A., 1995.
enzyme. On the other hand MCLR is an inhibitor of protein
Molecular cloning and functional expression of a recombinant
phosphatases what favors hyperphosphorilation, that is an
72.5 kDa fragment of the 110 kDa regulatory subunit of smooth
increased response to protein kinases. Protein kinase C
muscle protein phosphatase 1M. FEBS Lett. 377 (2), 123–127.
which is responsible for the activation of phospholipase
Hooser, S.B., Beasley, V.R., Lovell, R.A., Carmichael, W.W.,
A2 (Braquet, 1987) could be increased by the action of
Haschek, W.M., 1989. Toxicity of microcystin-LR, a cyclic
MCLR. The arachidonic acid inhibits L-type calcium
heptapeptide hepatotoxin from Microcystis aeruginosa, to rats
current, via a mechanism which involves, in part, stimula-
and mice. Vet. Pathol. 26, 246–252.
tion of protein phosphatase activity. On the other hand,
Kotak, B.G., Semalulu, S., Fritz, D.L., Prepas, E.E., Hrudey, S.E.,
dexamethasone and indomethacin inhibit arachidonic acid
Coppock, R.W., 1996. Hepatic and renal pathology of intraper-itoneally administered microcystin-LR in Rainbow Trout
metabolism, what favors an inhibition of activity in these
(Oncorhynchus Mykiss). Toxicon 34, 517–525.
protein (Petit-Jacques and Hartzell, 1996). With the use of
Kuroda, N., Hayashi, Y., Matozaki, T., Hanioka, K., Gotoh, A.,
indomethacin and dexamethasone we raise the possibility of
Wang, W., Uchida, H., Hashimoto, K., Iwai, Y., Kawasaki,
the participation of prostaglandin and of calcium, and phos-
K., Imai, Y., Kasuga, M., Itoh, H., 1998. Differential expression
pholipase A2, respectably, in the alterations triggered by
of SHP2, a protein-tyrosine phosphatase with SRC homology-2
domains, in various types of renal tumour. Virchows Arch. 433
According to the literature MCLR affects two types of cells:
hepatocytes and macrophages. In the hepatocytes the toxin
Naseem, S.M., Hines, H.B., Creasia, D., 1990. A inhibition of
acts as an inhibitor of protein phosphatases and an activator
microcystin-induced release of cyclooxygenase products from
rat hepatocytes by anti-inflammatory steroids. Proc. Soc. Exp.
the toxin induces TNFa and IL-1 (Rocha et al., 2000). Cyto-
Nishiitsutji-Uwo, G.M., Ross, B.D., Krebs, H.A., 1967. Metabolic
kines induce the production of PAF, and the activation of
activities of the isolated perfused rat kidney. Biochem. J. 103,
prostaglandin which seem to be involved in the shock caused
by MCLR. Microcystin specifically and potently inhibits
Nobre, A.C.L., Jorge, M.C.M., Menezes, D.B., Fonteles, M.C.,
protein phosphatase 1 and 2A. M110 and SHP2 that are
Monteiro, H.S.A., 1999. Effects of microcystin-LR in isolated
expressed in kidney. (Haystead et al., 1995; Kuroda et al.,
perfused rat kidney. Brazilian J. Med. Biol. Res. 32, 985–988. A.C.L. Nobre et al. / Toxicon 39 (2001) 721–724
Petit-Jacques, J., Hartzell, H.C., 1996. Effect of arachidonic acid on
Guerrant, R.L., Ribeiro, R.A., Lima, A.A.M., 2000. Superna-
the L-type calcium current in frog cardiac myocytes. J. Physiol.
tants from macrophages stimulated with microcystin-LR induce
electrogenic intestinal response in rabbit ileum. Pharmacology
Radbergh, C.M.I., Bylund, G., Eriksson, J.E., 1991. Histological
effects of microcystin-LR, a cyclic peptide toxin from the cyano-
Ross, B.D., 1978. The isolated perfused rat kidney. Clinical Sci.
bacterium (blue-green alga) Microcystis aeruginosa, on common
carp (Cyprinus carpio L.). Aquatic. Toxicol. 20, 131–146.
Walson, M., Davidson, D.G., Orloff, J., 1955. The renal clearance of
Rocha, M.F.G., Sidrim, J.J.C., Soares, A.M., Jimenez, G.C.,
alkali-stable inulin. J. Clin. Invest. 34, 1520–1523.
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