Toxicology Letiers, 53 (1990) 247-250
247
Elsevier TGXLET 02430
Protective effect of verapamil in cyclosporinincubated isolated rat glomeruli
Beatrice L. Azou and Jean Cambar Groupe d’EtuaS de Physiologic et Physiop~th~~og~ R&ales, Fact&
de Pharmacie, Bordeaux (France)
Key words: Cyclosporin: Isolated glomeruli; Vasoreactivity; Verapamil; Rat
INTRODUCTION
Cyclosporin (CsA) has a powerful immunomodulating activity producing an amelioration in short- and medium-term survival of transplanted organs. It is nevertheless able to induce dramatic side-effects, such as hypertension and nephrotoxicity. It has already been described as a chronic nephrotoxin [ 11,eTentially characterized by tubulo-interstitial lesions in kidney biopsies and by irreversible damage to renal function; moreover, an acute nephrotoxicity depending upon a different mechanism can also be observed [2,3]. This acute renal toxicity is characterized by a large decrease in renal blood flow (RBF), in glomerular filtration rate (GFR) and in glomerular ultrafiltration coefficient (&) 121;in additions, exposure to Csa is associated with a marked increase in renal vascular resistance (RVR) [4]. Isolated glomeruli from rat renal cortex without any vascular nervous or humoral influences, represent a useful model to assess a possible direct effect of endogenous [5] or exogenous [6] vasoactive substances. The aim of our study was to confirm the direct vasoactive effect of CsA in isolated glomeruli and to obtain evidence for a possible protective effect of verapamil in CsAincubated isolated rat glomeruli at various concentrations of this drug. MATERIALS AND METHODS
Glomeruli were isolated from kidneys removed from rats weighing 200 f 20 g after decapitation. The outer portion of the renal cortex containing superficial glomeruli Address for correspondence:
J. Cambar, Groupe d’ktude de Physiologie et Physiopathologie Rbnales, Facult6 de Pharmacie, 3 Place de la Victoire, 33000 Bordeaux, France. 0378-4274/~/$3.50
@ 1990 Elsevier Science Publishers B.V. (Biorn~i~~ Division)
248
was dissected and retained. Renal tissue was reduced to a homogenous pulp with scissors. Glomeruli were isolated by passing the pulp through calibrated sieves and suspended in Hank’s balanced saline solution at pH 7.4. The suspension was centrifuged at 1000 x g for 60 s in the same solution. The glomeruli obtained did not present their Bowman’s capsule; tubular contamination was less than 3 $ and no arterioles were observed in the preparation. Incubation with lOA M CsA (Sandimmun, Sandoz) was performed at room temperature because of its fair vasoconstrictive effect which decreased dramatically the glomerular area (1O-‘o M, -4.2%, lop9 M, -6.O& lop8 M, -10.3%; 1O-6 M, - 12.1%) after 10 min of incubation. Each glomerulus served as its own control, the same glomerulus being observed under a microscope (BH2, Olympus) and photographed after control of the loss of Bowman’s capsule. The glomerular planar surface area (average of 15-20 glomeruli) was quantified for each experimental condition (CsA alone or CsA with verapamil) after 0, 5, 10, 20 and 30 min of drug incubation, using a computerized image analyser (BIOCOM 200). All experiments were performed under double-blind conditions. The results were expressed as mean surface f SEM. Statistical analysis was performed by analysis of variance followed by Student’s test. Differences at the 5 % level or less were considered as statistically significant. RESULTS
Under toxic conditions (10 -6 M CsA alone), the reduction of glomerular size induced by CsA was rapid and time-dependent. The decrease was regular: - 2.1% at 5 min, -4.2% at 10 min, -9.1% at 20 min, and - 12.1% at 30 min. This vasoconstrictive response was highly significant compared with the control (unintoxicated glomeruli) (P < 0.001). In verapamil-pretreated CsA-intoxicated glomeruli, at all verapamil concentrations (lop5 to lop8 M) a significant decrease in glomerular size reduction was observed. But a better protective effect of verapamil could be noted at lop6 and lop7 M. Under these experimental conditions, after 30 min, the decrease in size was only - 1.0% for lop6 M verapamil and -3.7% for 10W7M (versus - 12.1% at 30 min with CsA alone). Thus a very highly significant difference in size change between verapamil-pretreated CsA-intoxicated glomeruli and only CsA-intoxicated glomeruli was observed. DISCUSSION
The different physiopathological mechanisms leading to CsA-induced acute nephrotoxicity are not yet clearly established. Various clinical and experimental data
249
agree with alterations of intrarenal hemodynamics [2-4]. Such a change in renal hemodynamics can be demonstrated in scanning electron microscopy by a progressive decrease in afferent arteriole calibre, reaching only 34% of control values after 2 weeks of CsA treatment [3,7]. The present observations demonstrate that the different CsA concentrations can induce a vasoconstrictive effect in isolated rat glomeruli. This direct vasoactive effect is dose- and time-related. Thus, CsA induced a very significant decrease in glomerular surface area (- 12.1% at 30 min for 10F6 M CsA). Such a decrease in glomerular area has been observed with different vasoconstrictive agents [8,9], which are able to induce renal hemodynamic changes in vivo. This contraction decreases renal blood flow and glomerular filtration rate by a reduction in filtration surface and Kf [8,10]. Moreover, recent studies 17,101have demonstrated that CsA induced a vasoconstriction of vascular beds involving glomerular afferent arterioles; these observations suggest a possible action of CsA also on glomerular capillaries. This experimental model permits us to eliminate possible renal nervous and hormonal interference. The present results show that CsA induces a direct vasoconstriction at the glomerular level. The exact mechanism of the vasoconstrictive effect of CsA remains to be clarified. These vas~onst~ctive effects observed in vitro could explain in part the dramatic hemodynamic changes and the development of acute renal toxicity induced in vivo by CsA [ 111. It was recently demonstrated in vivo in man [ 121 and in mice [13] that verapamil ameliorates acute CsA-induced nephrotoxicity by preventing blood flow impairment and a decrease in glomerular hypofusion secondary to vasoconstriction of the afferent glomerular arteriole. Vasodilators have recently been shown to attenuate the vasoconstrictive effect of CsA in isolated perfused rabbit kidney [14]. The present study confirms in vitro these data. Indeed, verapamil dramatically reduces the vasoconstrictive effect of CsA, glomerular size being reduced by - 12.1% with CsA alone and by only - 1.0% with 10m6 M verapamil. REFERENCES I Myers, B.D., Sibley, R., Newton, L., Tomlanovich, S.J., Boshkos, C., Stinson, E., Luetscher, J.A., Whitney, D.J.. Krasny, D., Coplon, N.S. and Perlroth, M.G. (1988) The long-term course of cyclosporin-associated chronic nephropathy. Kidney Int. 33,590-600. 2 Barros, E.J.G., Boim, M.A., Ajzen, H., Ramos, O.L. and Schor, N. (1987) Glomerular hemodynami~s and hormonal participation on cyclosporin nephrotoxicity. Kidney Int. 32,19X5. 3 English, J.. Evan, A., Houghton, D.C. and Bennett, W. (1987) Cyclosporin-induced acute renal dysfunction in the rat. Transplant. Proc. 44, 135-141. 4 Curtis, J.J., Luke, R.G., Dubovsky, E., Whelchel, J.D., Diethelm, A.G. and Jones, P. (1986) Cyclosporin in therapeutic doses increases renal allograft vascular resistance. Lancet 2,477479. 5 Ardaillou, R., Sraer, J., Chansel, D., Ardaillou, N. and Sraer, J.D. (1987) The effects of angiotensin II on isolated glomeruli and cultured glomerular cells. Kidney Int. 31,574580.
250 6 Cambar, J., Saurel, J. and Canellas, J. (1978) The myorelaxant effect of theophylline on smooth muscle cells of the afferent arterioles and glomerular tuft. Eur. J. Pharmacol. 49, 193-196. 7 Xue, H., Bukoski, R.D., McCarron, D.A. and Bennett, W. (1987) Induction of contraction in isolated rat aorta by cyclosporin. Transplant. Proc. 43,715-718. 8 Dworkin, L.D., Ichikawa, 1. and Brenner, B.M. (1983) Hormonal modulation of glomerular function. Am. J. Physiol. 244, F95-F104. 9 Kreisberg, J.I., Venka~chalam, M. and Troyer, D. (1985) Contractile properties of cultured f$omerular mesangial cells. Am. J. Physiol. 249, F457-F463. 10 Schor, N., Ichikawa, I. and Brenner, B.M. (1981) Mechanism of action of various hormones and vasoactive substances on glomerular ultraliltration in the rat. Kidney Int. 20,442-451. 11 Dieperink, H., Leyssac, P.P., Kemp, E., Starklint, H., Frandsen, N,E. and Tvedes, N. (1987) Nephrotoxicity of cyclosporin A in humans: effects on glomerular filtration and tubular reabsorption rates. Eur. J. Clin. Invest. 17,493496. 12 Dawidson, I., Rooth, P., Fisher, D., Fry, W.R., Alway, C., Coorpender, L. and Reisch, J. (1989) Verapamil ameliorates acute cyclosporin A (CsA) nephrotoxicity and improves immunosuppression after cadaver renal transplantation. Transplant Proc. 21, 151l-1513. 13 Rooth, P., Dawidson, I., Diller, K. and Taljedal, I. (1988) Protection against cyclosporin-induced impairment of renal microcirculation by verapamil in mice. Transplant. Proc. 45433-437. 14 Cairns, H.S., Fairbanks, L.D., Westwick, J. and Neild, G.H. (1989) Cyclosporin therapy in vivo attenuates the response to vasodilators in the isolated perfused kidney of the rabbit. Br. J. Pharmacol. 98, 463-468.
247
Elsevier TGXLET 02430
Protective effect of verapamil in cyclosporinincubated isolated rat glomeruli
Beatrice L. Azou and Jean Cambar Groupe d’EtuaS de Physiologic et Physiop~th~~og~ R&ales, Fact&
de Pharmacie, Bordeaux (France)
Key words: Cyclosporin: Isolated glomeruli; Vasoreactivity; Verapamil; Rat
INTRODUCTION
Cyclosporin (CsA) has a powerful immunomodulating activity producing an amelioration in short- and medium-term survival of transplanted organs. It is nevertheless able to induce dramatic side-effects, such as hypertension and nephrotoxicity. It has already been described as a chronic nephrotoxin [ 11,eTentially characterized by tubulo-interstitial lesions in kidney biopsies and by irreversible damage to renal function; moreover, an acute nephrotoxicity depending upon a different mechanism can also be observed [2,3]. This acute renal toxicity is characterized by a large decrease in renal blood flow (RBF), in glomerular filtration rate (GFR) and in glomerular ultrafiltration coefficient (&) 121;in additions, exposure to Csa is associated with a marked increase in renal vascular resistance (RVR) [4]. Isolated glomeruli from rat renal cortex without any vascular nervous or humoral influences, represent a useful model to assess a possible direct effect of endogenous [5] or exogenous [6] vasoactive substances. The aim of our study was to confirm the direct vasoactive effect of CsA in isolated glomeruli and to obtain evidence for a possible protective effect of verapamil in CsAincubated isolated rat glomeruli at various concentrations of this drug. MATERIALS AND METHODS
Glomeruli were isolated from kidneys removed from rats weighing 200 f 20 g after decapitation. The outer portion of the renal cortex containing superficial glomeruli Address for correspondence:
J. Cambar, Groupe d’ktude de Physiologie et Physiopathologie Rbnales, Facult6 de Pharmacie, 3 Place de la Victoire, 33000 Bordeaux, France. 0378-4274/~/$3.50
@ 1990 Elsevier Science Publishers B.V. (Biorn~i~~ Division)
248
was dissected and retained. Renal tissue was reduced to a homogenous pulp with scissors. Glomeruli were isolated by passing the pulp through calibrated sieves and suspended in Hank’s balanced saline solution at pH 7.4. The suspension was centrifuged at 1000 x g for 60 s in the same solution. The glomeruli obtained did not present their Bowman’s capsule; tubular contamination was less than 3 $ and no arterioles were observed in the preparation. Incubation with lOA M CsA (Sandimmun, Sandoz) was performed at room temperature because of its fair vasoconstrictive effect which decreased dramatically the glomerular area (1O-‘o M, -4.2%, lop9 M, -6.O& lop8 M, -10.3%; 1O-6 M, - 12.1%) after 10 min of incubation. Each glomerulus served as its own control, the same glomerulus being observed under a microscope (BH2, Olympus) and photographed after control of the loss of Bowman’s capsule. The glomerular planar surface area (average of 15-20 glomeruli) was quantified for each experimental condition (CsA alone or CsA with verapamil) after 0, 5, 10, 20 and 30 min of drug incubation, using a computerized image analyser (BIOCOM 200). All experiments were performed under double-blind conditions. The results were expressed as mean surface f SEM. Statistical analysis was performed by analysis of variance followed by Student’s test. Differences at the 5 % level or less were considered as statistically significant. RESULTS
Under toxic conditions (10 -6 M CsA alone), the reduction of glomerular size induced by CsA was rapid and time-dependent. The decrease was regular: - 2.1% at 5 min, -4.2% at 10 min, -9.1% at 20 min, and - 12.1% at 30 min. This vasoconstrictive response was highly significant compared with the control (unintoxicated glomeruli) (P < 0.001). In verapamil-pretreated CsA-intoxicated glomeruli, at all verapamil concentrations (lop5 to lop8 M) a significant decrease in glomerular size reduction was observed. But a better protective effect of verapamil could be noted at lop6 and lop7 M. Under these experimental conditions, after 30 min, the decrease in size was only - 1.0% for lop6 M verapamil and -3.7% for 10W7M (versus - 12.1% at 30 min with CsA alone). Thus a very highly significant difference in size change between verapamil-pretreated CsA-intoxicated glomeruli and only CsA-intoxicated glomeruli was observed. DISCUSSION
The different physiopathological mechanisms leading to CsA-induced acute nephrotoxicity are not yet clearly established. Various clinical and experimental data
249
agree with alterations of intrarenal hemodynamics [2-4]. Such a change in renal hemodynamics can be demonstrated in scanning electron microscopy by a progressive decrease in afferent arteriole calibre, reaching only 34% of control values after 2 weeks of CsA treatment [3,7]. The present observations demonstrate that the different CsA concentrations can induce a vasoconstrictive effect in isolated rat glomeruli. This direct vasoactive effect is dose- and time-related. Thus, CsA induced a very significant decrease in glomerular surface area (- 12.1% at 30 min for 10F6 M CsA). Such a decrease in glomerular area has been observed with different vasoconstrictive agents [8,9], which are able to induce renal hemodynamic changes in vivo. This contraction decreases renal blood flow and glomerular filtration rate by a reduction in filtration surface and Kf [8,10]. Moreover, recent studies 17,101have demonstrated that CsA induced a vasoconstriction of vascular beds involving glomerular afferent arterioles; these observations suggest a possible action of CsA also on glomerular capillaries. This experimental model permits us to eliminate possible renal nervous and hormonal interference. The present results show that CsA induces a direct vasoconstriction at the glomerular level. The exact mechanism of the vasoconstrictive effect of CsA remains to be clarified. These vas~onst~ctive effects observed in vitro could explain in part the dramatic hemodynamic changes and the development of acute renal toxicity induced in vivo by CsA [ 111. It was recently demonstrated in vivo in man [ 121 and in mice [13] that verapamil ameliorates acute CsA-induced nephrotoxicity by preventing blood flow impairment and a decrease in glomerular hypofusion secondary to vasoconstriction of the afferent glomerular arteriole. Vasodilators have recently been shown to attenuate the vasoconstrictive effect of CsA in isolated perfused rabbit kidney [14]. The present study confirms in vitro these data. Indeed, verapamil dramatically reduces the vasoconstrictive effect of CsA, glomerular size being reduced by - 12.1% with CsA alone and by only - 1.0% with 10m6 M verapamil. REFERENCES I Myers, B.D., Sibley, R., Newton, L., Tomlanovich, S.J., Boshkos, C., Stinson, E., Luetscher, J.A., Whitney, D.J.. Krasny, D., Coplon, N.S. and Perlroth, M.G. (1988) The long-term course of cyclosporin-associated chronic nephropathy. Kidney Int. 33,590-600. 2 Barros, E.J.G., Boim, M.A., Ajzen, H., Ramos, O.L. and Schor, N. (1987) Glomerular hemodynami~s and hormonal participation on cyclosporin nephrotoxicity. Kidney Int. 32,19X5. 3 English, J.. Evan, A., Houghton, D.C. and Bennett, W. (1987) Cyclosporin-induced acute renal dysfunction in the rat. Transplant. Proc. 44, 135-141. 4 Curtis, J.J., Luke, R.G., Dubovsky, E., Whelchel, J.D., Diethelm, A.G. and Jones, P. (1986) Cyclosporin in therapeutic doses increases renal allograft vascular resistance. Lancet 2,477479. 5 Ardaillou, R., Sraer, J., Chansel, D., Ardaillou, N. and Sraer, J.D. (1987) The effects of angiotensin II on isolated glomeruli and cultured glomerular cells. Kidney Int. 31,574580.
250 6 Cambar, J., Saurel, J. and Canellas, J. (1978) The myorelaxant effect of theophylline on smooth muscle cells of the afferent arterioles and glomerular tuft. Eur. J. Pharmacol. 49, 193-196. 7 Xue, H., Bukoski, R.D., McCarron, D.A. and Bennett, W. (1987) Induction of contraction in isolated rat aorta by cyclosporin. Transplant. Proc. 43,715-718. 8 Dworkin, L.D., Ichikawa, 1. and Brenner, B.M. (1983) Hormonal modulation of glomerular function. Am. J. Physiol. 244, F95-F104. 9 Kreisberg, J.I., Venka~chalam, M. and Troyer, D. (1985) Contractile properties of cultured f$omerular mesangial cells. Am. J. Physiol. 249, F457-F463. 10 Schor, N., Ichikawa, I. and Brenner, B.M. (1981) Mechanism of action of various hormones and vasoactive substances on glomerular ultraliltration in the rat. Kidney Int. 20,442-451. 11 Dieperink, H., Leyssac, P.P., Kemp, E., Starklint, H., Frandsen, N,E. and Tvedes, N. (1987) Nephrotoxicity of cyclosporin A in humans: effects on glomerular filtration and tubular reabsorption rates. Eur. J. Clin. Invest. 17,493496. 12 Dawidson, I., Rooth, P., Fisher, D., Fry, W.R., Alway, C., Coorpender, L. and Reisch, J. (1989) Verapamil ameliorates acute cyclosporin A (CsA) nephrotoxicity and improves immunosuppression after cadaver renal transplantation. Transplant Proc. 21, 151l-1513. 13 Rooth, P., Dawidson, I., Diller, K. and Taljedal, I. (1988) Protection against cyclosporin-induced impairment of renal microcirculation by verapamil in mice. Transplant. Proc. 45433-437. 14 Cairns, H.S., Fairbanks, L.D., Westwick, J. and Neild, G.H. (1989) Cyclosporin therapy in vivo attenuates the response to vasodilators in the isolated perfused kidney of the rabbit. Br. J. Pharmacol. 98, 463-468.
