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Calcium Channel Blocker Nisoldipine Limits Ischemic Damage in Rat Kidney
0022-5347 /85/1346-1251$02.00/0 Voi. 134, Decembe,Printed in U.S.A.
THE .JOURNAL OF UROLOGY
Copyright([:) 1985 by The Williams & Wiikins Co.
CALCIUM CHANNEL BLOCKER NISOLDIPINE LIMITS ISCHEMIC DAMAGE IN RAT KIDNEY LOTHAR HERTLE* AND BERNWARD GARTH OFF From the Department of Urology, University of Bochum and the Institute of Pharmacology, Bayer AG, Wuppertal, Federal Republic of Germany
ABSTRACT
The effects of the calcium channel blocker nisoldipine on renal function after 60 min. normothermic ischemia and contralateral nephrectomy were studied in male Wistar rats. Nisoldipine (300 ppm) was given in a standard diet as well as one hour prior to ischemia (10 mg./kg. orally). Survival, serum urea, serum creatinine, urine volume and creatinine clearance were used to test the effectiveness of the drug. Nisoldipine treatment resulted in the survival of all animals (compared to 66.6 per cent in the untreated group) and improved immediate and long term (14 days) renal function. The drug given post ischemia only was not effective, suggesting that nisoldipine must be present in the kidney during ischemia. The beneficial effects of the drug in postischemic acute renal failure may be attributed in part to effects on postischemic renal hemodynamics. Additional direct effects on ischemic renal epithelial cells, presumably by inhibiting transmembrane calcium fluxes, cannot be excluded. Without an effective protective therapy, reperfusion of the kidney after prolonged periods of normothermic ischemia causes extensive and often irreversible damage. Although the nature of this damage has been well documented,1 its precise cause remains obscure. 2 Recently, considerable interest has been focused on the role of calcium as a possible mediator of irreversible cell damage following a period of ischemia. Anoxic-ischemic injury of cell membranes is associated with a precipitous influx of calcium from the extracellular to the intracellular compartment, and, as a consequence, with the activation of calcium-dependent catabolic processes. 3 An analysis of this process has led to the suggestion that calcium channel blockers may prevent metabolic disturbances and promote functional and structural recovery after ischemia or anoxia. 4 Several experimental studies on myocardial, 4 • 5 hepatic 6 and cerebraF ischemia support this conjecture. Recent results8 • 9 suggest that impaired mitochondrial oxygen uptake and increased cytosolic calcium concentration may be important events also in the pathogenesis of postischemic and toxic acute renal failure (ARF). Recent papers contain conflicting reports regarding the effects of the calcium channel blocker verapamil on postischemic ARF. Some authors observed beneficial effects of the drug in norepinephrine-induced ARF 10 • 11 as well as in clamping models of ARF 12- 14 and others observed no effects in clamping models of postischemic ARF .11 • 15 Although the calcium channel blocking drugs share the ability to competitively inhibit transmembrane calcium fluxes in smooth and cardiac muscle, 16 it has been noted that they are structurally dissimilar, possessing diverse pharmacological properties. Nisoldipine, a new dihydropyridine derivative (structure formula in fig. 1) chemically similar to nifedipine, exerts potent relaxant effects on vascular smooth muscle. Experimentally, nisoldipine has demonstrated favorable effects on ischemic myocardium in the intact animal. 17 The drug also has an antihypertensive effect in various models of experimental hypertension. 17 The present study was undertaken to investigate whether nisoldipine could protect the rat kidney against ischemic damage. A further aim of the study was to evaluate if nisoldipine Accepted for publication July 10, 1985. * Requests for reprints: Urologische Klinik, Ruhr-Universitiit Bochum, Klinikum Marienhospital, Widumer Str. 8, D-4690 Herne 1, Federal Republic of Germany.
could provide additional protective effects in hypothermic ischemia, which might be of clinical relevance in renal surgery and transplantation. MATERIALS AND METHODS
All experiments were performed on male Wistar rats weighing 180 to 220 gm. The animals were housed in individual metabolic cages and maintained on tap water and a standard pellet diet (ssniff-Versuchstier-Alleindiat), to which they had free access pre- and postoperatively. Four days prior to the study, the right kidney was removed through a small flank incision under ether anesthesia. For preparation of the left kidney, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, Abbott) 40 mg./ kg. body weight (b.w.) and placed on a heated table that maintained body temperature between 37C and 38C. The kidney was exposed by a flank incision, freed from perirenal fat and left attached only by its pedicle. Temporary renal ischemia was induced by occlusion of the vascular pedicle with a microvascular clamp (Y asargil aneurysm clip, Aesculap, closing force 0.39 to 0.49 N) for 60 min. In sham operated animals, the kidneys were prepared identically except for clamping the vascular pedicle. The flank was left exposed for a full 60 min. In the rats subjected to hypothermic ischemia, frozen 5 per cent dextrose slush was placed around the kidney during occlusion of the renal pedicle. After completion of surgery and recovery from anesthesia the rats were returned to their cages. Experiments were performed on seven groups as described in table 1. In groups II, IV and VII nisoldipine (Bayer AG) was given in the standard diet four days prior to, and during the three or fourteen day observation period after ischemia. The concentration of the drug in the diet was 300 ppm (mg./kg. diet). Additionally, nisoldipine (10 mg./kg. b.w.) was administered one hour before induction of ischemia by gavage. In group V the drug was given by gavage one hour after declamping (10 mg./kg. b.w.) and in the diet (300 ppm) during the three days observation period after ischemia. Groups III and IV were subdivided, 10 animals of each group observed over a period of fourteen days and the others for three days. Groups I and II were observed for fourteen days after ischemia, and animals of groups V to VII for three days. Animals were examined daily and weighed at regular intervals. Serum creatinine and urea were determined just prior to ischemia (day 0) and on days 1 and 3 in all groups and further
1251
1252
HERTLE AND GARTHOFF
third of the animals being totally anuric on the first day after declamping. Most of these animals died two to six days after the ischemic insult, and in all cases deaths were due to uremia. All animals of this group had raised serum urea and creatinine concentrations and a diminished creatinine clearance on the first, third and seventh day after ischemia and the values had not returned to normal 14 days after ischemia. The peak rise in urea and creatinine occurred on day 3 after ischemia. By day 3 the surviving animals excreted large volumes of dilute urine, more than 200 per cent of the preischemic values (day 7: 27.2 ± 1.9 µl./min. and day 14: 20.5 ± 2.9 µ1./min.; no.= 6). All animals treated with nisoldipine prior and subsequent to normothermic ischemia (group IV) survived the insult (significantly different versus group III on day 3, p <0.0005). Rats of
on days 7 and 14 in groups I to IV after ischemia. On the same days as indicated above, 24 hr. urine samples were collected. Urine volume and creatinine levels were determined. Endogenous creatinine clearance was estimated from the total 24 hr. creatinine excretion and from the serum creatinine value. Improved survival was tested using Fisher's exact test. 18 The remaining data from the different groups or subgroups 1 or 3 days after ischemia were subjected to analysis of variance. This procedure was followed by Dunnett's test19 or Scheffe's test19 to identify the sources of differences; in cases where varianceinstability was detected data were subjected to logarithmic transformation. A p-value of less than 0.05 was considered to be significant. Results are expressed as means ± standard error of the means (SEM), if not differently indicated. RESULTS
Control values before ischemia (day 0) for all tested parameters (mean values ± standard deviations; no. = 110) were: serum urea 9.2 ± 1.6 mmol./1., serum creatinine 62 ± 15 µmol./ 1., urine volume 3. 7 ± 1.6 µ1./min. and endogenous creatinine clearance 518.3 ± 175.7 µl./min. Figure 2 and tables 2-4 summarize the renal function changes following sham operation or ischemia in the 7 groups of animals. Sham operation without and with nisoldipine treatment (groups I and II) had no detrimental effects on renal excretory function over a fourteen day observation period. Rats subjected to 60 min. normothermic ischemia without any protection (group III) developed an ARF with about one
mmol/1 7
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40
20
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9
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----~ 10A # -!12=--/...!:!=o_ ,/•
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days after
14 ischemia or sham operation
FIG. 2. Serum urea concentrations in uninephrectomized rats after sham operation or 60 min. of normothermic ischemia: A sham-operated, untreated (group I), b, sham-operated, nisoldipine treated (group II),• ischemia, untreated (subgroup III), 0 ischemia, nisoldipine treated (subgroup IV). Means ± SEM; number of surviving animals given at symbols. Day 0: values before sham operation or ischemia. Raised serum urea levels throughout observation period in ischemia-untreated group with considerable mortality. No mortality and lower urea levels at all days after ischemia in nisoldipine-treated group. For statistical evaluation refer to tables 3 and 4.
FIG. 1. Chemical structure of nisoldipine (Isobutyl methyl 1,4-dihydro-2,6-diethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate). TABLE
Group
1. Characteristics of experimental groups
9
33 (10/23) 28 (10/18)
III IV V
10
VI VII
10 10
14 14
Sham-operated, untreated Sham-operated, nisoldipine treated (prior and subsequent to sham operation) N ormothermic ischemia, untreated Normothermic ischemia, nisoldipine treated (prior and subsequent to ischemia) N ormothermic ischemia, nisoldipine treated (only subsequent to ischemia) Hypothermic ischemia, untreated Hypothermic ischemia, nisoldipine treated (prior and subsequent to ischemia)
10
I II
Recovery Period (Days)
Protocol.
No.
14/3 14/3 3 3
3
Groups III and IV were subdivided, with 10 animals of each group followed 14 days and 23 animals of group III and 18 animals of group IV followed 3 days after ischemia.
TABLE 2.
Serum creatinine and creatinine clearance after sham operation or 60 min. normothermic ischemia followed over 14 days. Number of surviving animals in brackets. Means ± SEM
I II III IV
(no.= (no.= (no.= (no.=
Creatinine Clearance (µ!./min.)
Serum Creatinine (µmol./1.)
Experimental Group
1
Day 10) 9) 10) 10)
57 ± 56 ± 274 ± 170 ±
2 (10) 2 (9) 23 (9) 15 (10)
For statistical evaluation refer to tables 3 and 4.
3 63 54 302 126
± ± ± ±
7 (10) 1 (9) 33 (7) 31 (10)
7 57 61 171 68
± ± ± ±
2 (10) 6 (9) 18 (6) 2 (10)
14 61 ± 66 ± 104 ± 56 ±
2 (10) 5 (9) 13 (6) 1 (10)
1 610 ± 547 ± 49 ± 144 ±
3 26 29 12 17
609 465 111 338
± ± ± ±
7 70 66 19 44
541 ± 425 ± 199 ± 454 ±
14 46 31 43 38
581 477 425 563
± ± ± ±
40 43 92 52
TABLE
3. .Suruiual, serum creatinine (8Cr), serum urea (SUr), urine volume (U Vol) and creatinine clearance (CrCl) on the first day afte:~ sham
operation or ischemia. _Means ± SEJvf Experimental Group
Survival
I II III
100 100 96.9 100 100 100 100
(%)
IV V VJ Vil
SCr (µmol./1.) 57 56 259 196 300 63 53
±
28**
± ± ± ± ±
2'** 11 14"** 16 2"** ± 3ab**
SUr (mmol./1.) 8.6 8.5 45.1 35.2 47.3 12.0 9.6
± ± ± ± ± ± ±
0.2"** 0.3"** 2.0 2.4"* 2.0 0.6"** o.4•b**
UVol (µ!./min.) 4.4 5.2 5.3 11.1 7.7 6.4 8.2
± ± ± ± ± ± ±
0.4 0.6 0.8 1.1"** 1.5 0.7 0.6
CrCl (µ!./min.) 610.4 54 7 .3 49.7 132.1 40.2 594.0 733.4
± ± ± ± ± ± ±
25.6"** 28. 7"** 10.8 17.1'** 7.4 61.0"** 67.4"**
• Significantly different from group III (corresponding subgroups). b Values of group VJ! significantly different from those of group VI. * p <0.05, ** p <0.01.
TABLE 4.
Survival, serum creatinine (SCr), serum urea (SUr), urine volume (U Vol) and creatinine clearance (CrCl) on third day after sham operation or ischemia. Means ± SEM
Experimental Group
Survival (%)
I
100 100 66.6 100 90 100 100
II III IV V VI VII
SCr (µmol./1.) 63 ± 54± 280 ± 201 ± 264 ± 59 ± 56 ±
7"** la** 23 21 "** 50 3"** 2"**
SUr (mmol./1.) 8.6 9.1 63.7 41.6 57.1 12.0 10.4
± ± ± ± ± ± ±
0.3"** 0.4"** 4.4 4.8"** 5.3 0.5"** 0.3"**
UVol (µ!./min.) 3.7 2.9 13.0 13.3 13.5 7.5 9.3
± ± ± ± ± ± ±
0.3"** 0.7"** 1.5 0.2 1.6 1.0"** 1.0'*
CrCl (µ!./min.) 608.6 464.6 122.2 224. 7 161.2 749.8 831.3
± ± ± ± ± ± ±
69.6"** 66.2"** 20.0 28.9"** 40.9 62.4"** 52.6"**
• Significantly different from group III (corresponding subgroups). * p <0.05, p <0.01.
group IV had significantly lower serum urea, serum creatinine, and higher creatinine clearance on the first and third day after ischemia than the untreated rats (group Animals treated with nisoldipine had a significantly higher urine volume on day 1 after ischemia than the untreated group. On days 7 and 14 after ischemia the nisoldipine treated animals had normal serum urea, serum creatinine and creatinine clearance values, whereas these values were still abnormal in the untreated rats, The severity of ARF in rats in which nisoldipine administration started after declamping (group V) was similar to that of the untreated animals, Rats treated with regional hypothermia alone (group VI) had slightly elevated serum urea values and urine volumes on days 1 and 3 after ischemia, The values of serum creatinine and creatinine clearance on days 1 and 3 after ischemia did not differ from those on day 0, Animals treated with hypothermia in combination with nisoldipine (group VII) had significantly lower serum creatinine and urea concentrations on day 1 after ischemia with group VL All animals of groups VI and VII the insult, DISCUSSION
A period of 60 min, normothermic ischemia after contralateral nephrectomy resulted in a severe and sometimes lethal renal Nisoldipine treatment before inducing w,,rnorn,w. as well as continuing treatment in the period thereafter, was found to be highly effective in preventing uremic death and improving immediate and long term days) renal function. However, when the drug was given after ischemia only, it was not effective, suggesting that nisoldipine must be present in the organ during the ischemic period if the injury is to be reduced, Regional hypothermia had a superior protective effect compared with nisoldipine, Treatment with nisoldipine in addition to external cooling provided only a slight benefit compared to hypothermia alone. The small size of rat kidney, however, does not allow a definite statement on a possible superior protective effect in larger kidneys treated with the calcium channel blocker in combination with external cooling. The beneficial effects of calcium channel blockers on ischemically damaged tissues have been attributed to the blockade of deleterious calcium influx. 4 Although the cellular and molecular mechanisms leading to ischemic cell damage have not been
clarified, substantial evidence exists that the influx of calcium a majo:r role in mediating cellular injury, 3 Schanne et al. 20 showed that a range of membrane-active toxins only caused toxic cell death in the presence of normal extracellular calcium activity, When the extracellular calcium activity was reduced to that of normal intracellular activity, the toxins had little effect. The cell membrane separates extracellular millimolar calcium concentrations from intracellular micromolar calcium concentrations. This very large electrochemical gradient is maintained by the passive permeability of the plasma membranes to calcium ions and by active extrusion of calcium. If the membrane is damaged by ischemia or toxins, calcium readily enters and accumulates within the cell and results in the rapid death of the cell. 3 Elevated cytosolic calcium levels may have many detrimental effects on the cell: activation of calciumstimulated ATP-ase processes which will further deplete the cell of already stressed stores of ATP, activation of membranebound phospholipases resulting in alterations in membranebound enzyme and membrane permeability and increased free fatty acids which may act as detergents. 21 The exact mechanisms that initiate or maintain ARF are not yet clear. Numerous studies have indicated an association bein ca,cOc,~,c Of renal hemodynamics and tween major ARF, 2 However, exact role of cnam,re~ in renal blood flow in ARF continues to be controversial, since in experimental ARF renal blood flow may have returned to normal and yet the ARF persists as assessed by a continued marked reduction in glomerular filtration rate. 2 This disparity in the ratio of renal blood flow to glomerular filtration rate has suggested to many workers that other pathogenetic factors, such as tubular fluid back-leak and tubular obstruction, may play more important roles than a reduction in renal blood flow in various forms of ARF. 2 Venkatachalam et al. 1 have provided evidence that cytoplasmic blebs may constitute the substrate for intraluminal obstruction in ARF, The biochemical abnormalities which may initiate cellular death during renal ischemia, thereby providing substrate for tubular obstruction, have not been defined, Burke and Schrier8 showed that in renal ischemia a progressive increase in mitochondrial calcium accumulation occurs during reflow. These authors stated that an increased cytosolic calcium in epithelial '.,HaU,",vC
1254
HERTLE AND GARTHOFF
cells contributes to cell necrosis, and that this cellular debris provides the substrate for tubular obstruction and the maintenance of ARF. 22 Also, the initial ischemic phase of ARF may involve increased cytosolic calcium in the smooth muscle cells of the afferent arterioles, a phenomenon known to be associated with vasoconstriction. 23 Nisoldipine has potent relaxant effects on vascular smooth muscle. The EC 50 of the drug (the concentration eliciting halfmaximum relaxation) in potassium-induced contractions of isolated rabbit aortic strips is 2.4 X 10-9 mol./1. 17 Thus the beneficial effects of the drug in post-ischemic ARF may be attributed in part to effects on post-ischemic renal hemodynamics. However, attempts to overcome ischemic vasoconstriction directly by intra-renal artery administration of vasodilators and indirectly by the acute suppression of the renin-angiotensin system have generally been unsuccessful. 24 Furthermore, in the same experimental setting we found no benefit from the potent vasodilator minoxidil (unpublished data), suggesting that nisoldipine may have additional "calcium antagonistic" effects on tubular cells. These effects may include inhibition of deleterious calcium overload of ischemically damaged renal epithelial cells, although calcium channels have not been demonstrated to exist in renal tubular cells up to now. Although the results obtained in this study should be extrapolated with caution to the clinical situation, there is increasing appreciation of the potential of pharmacological calcium channel blockade in renal ischemia. Recently, it was reported25 that the incidence of ARF after cardiovascular surgery dropped after calcium channel blockers were used pre- and intraoperatively to improve cardiac function. It would seem that these drugs had an unexpected but decidedly beneficial side effect.
8. 9. 10.
11. 12.
13. 14.
15.
16. 17.
REFERENCES
1. Venkatachalam, M.A., Bernard, D. B., Donohoe, J. F. and Levinsky, N. G.: Ischemic damage and repair in the rat proximal tubule. Differences among the S,, 8 2 and 8 3 segments. Kidney Int., 14: 31, 1978. 2. Schrier, R. W., Burke, T. J., Conger, J. D. and Arnold, P. E.: Newer aspects of acute renal failure. Proc. 8th Int. Congr. Nephrol., Athens, pp. 63-69, 1981. 3. Farber, J. L.: Minireview. The role of calcium in cell death. Life Sciences, 29: 1289, 1981. 4. Fleckenstein, A.: Prevention by calcium antagonists of deleterious calcium overload: a new principle of cardioprotection. In: Calcium Antagonism in Heart and Smooth Muscle. Edited by A. Fleckenstein. New York, John Wiley & Sons, chapt. 3, pp. 109164, 1983. 5. Nayler, W. G.: The role of calcium in myocardial ischemia and cell death. In: Calcium Channel Blocking Agents in the Treatment of Cardiovascular Disorders. Edited by P. H. Stone and E. M. Antman. Mount Kisco, New York, Futura Publishing Co., pp. 81-105, 1983. 6. Peck, R. C. and Lefer, A. M.: Protective effect of nifedipine in the hypoxic perfused cat liver. Agents and Actions, 11: 421, 1981. 7. Hoffmeister, F., Kazda, S. and Krause, H. P.: Influence of nimo-
18. 19. 20. 21. 22.
23. 24. 25.
dipine (BAY e 9736) on the postischemic changes of brain function. In: Cerebral Blood Flow and Metabolism. Edited by F. Gotoh, H. Nagai and Y. Tazaki. Acta Neural. Scand., 60 (Suppl 72): 358, 1979. Burke, T. J. and Schrier, R. W.: Ischemic acute renal failure. Pathogenetic steps leading to acute tubular necrosis. Circulatory Shock, 11: 255, 1983. Weinberg, J.M., Harding, P. G. and Humes, H. D.: Alterations in renal cortex cation homeostasis during mercuric chloride and gentamicin nephrotoxicity. Exp. Mo!. Pathol., 39: 43, 1983. Burke, T. J., Arnold, P. E., Crossfield, P. D. and Schrier, R. W.: Effect of calcium membrane inhibition on norepinephrine-induced acute renal failure. In: Acute Renal Failure. Edited by H. E. Eliahou. London, John Libbey, pp. 239-240, 1982. Malis, C. D., Cheung, J. Y., Leaf, A. and Bonventre, J. V.: Effects of verapamil in models of ischemic acute renal failure in the rat. Am. J. Physiol., 245: F 735, 1983. Goldfarb, D., Iaina, A., Serban, I., Gavenda S., Kapuler, S. and Eliahou, H. E.: Beneficial effect of verapamil in ischemic acute renal failure in the rat. Proc. Soc. Exp. Biol. Med., 172: 389, 1983. Wait, R. B., White, P. G. and Davis, J. H.: Beneficial effects of verapainil on postischemic renal failure. Surgery, 94: 276, 1983. Kramer, H. J., Neumark, A., Schmidt, S., Klingmiiller, D. and Glanzer, K.: Renal functional and metabolic studies on the role of preventive measures in experimental acute ischemic renal failure. Clin. Exper. Dialysis and Apheresis, 7: 77, 1983. Blank, W., Unni Mooppan, M. M., Chhajwani, B., Chou, S. -Y. and Kim, H.: Effects of verapamil on preservation of renal function after ischemia: functional and ultrastructural study. J. Urol., 131: 992, 1984. Fleckenstein, A.: Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Ann. Rev. Pharmacol. Toxicol., 17: 149, 1977. Kazda, S., Garthoff, B., Riimsch, K. -D. and Schluter, G.: Nisoldipine. In: New Drugs Annual: Cardiovascular Drugs. Edited by A. Scriabine. New York, Raven Press, pp. 243-258, 1983. Sachs, L.: Angewandte Statistik, 6th Ed. Heidelberg, Springer Verlag, pp. 288-290, 1983. Wallenstein, S., Zucker, C. L. and Fleiss, J. S.: Some statistical methods useful in circulation research. Circ. Res., 47: 1, 1980. Schanne, F. A. X., Kane, A. B., Young, E. E. and Farber, J. L.: Calcium dependence of toxic cell death: a final common pathway. Science, 206: 700, 1979. Jennings, R. B.: Calcium ions in ischemia. In: Calcium Antagonists and Cardiovascular Disease. Edited by L. H. Opie. New York, Raven Press, pp. 85-95, 1984. Schrier, R. W., Arnold, P. E. and Burke, T. J.: Alterations in mitochondrial respiration and calcium movements in norepinephrine-induced acute renal failure: modification by mannitol. In: Acute Renal Failure. Edited by H. E. Eliahou. London, John Libbey, pp. 21-22, 1982. Van Neuten, J.M. and Vanhoutte, P. M.: Improvement of tissue perfusion with inhibitors of calcium ion influx. Biochem. Pharmacol., 29: 479, 1980. Eliahou, H. E.: Acute renal failure. In: Acute Renal Failure. Edited by H. E. Eliahou. London, John Libbey, pp. 1-5, 1982. Editorial: Promising agents limiting renal damage. JAMA, 249: 1987, 1983.
THE .JOURNAL OF UROLOGY
Copyright([:) 1985 by The Williams & Wiikins Co.
CALCIUM CHANNEL BLOCKER NISOLDIPINE LIMITS ISCHEMIC DAMAGE IN RAT KIDNEY LOTHAR HERTLE* AND BERNWARD GARTH OFF From the Department of Urology, University of Bochum and the Institute of Pharmacology, Bayer AG, Wuppertal, Federal Republic of Germany
ABSTRACT
The effects of the calcium channel blocker nisoldipine on renal function after 60 min. normothermic ischemia and contralateral nephrectomy were studied in male Wistar rats. Nisoldipine (300 ppm) was given in a standard diet as well as one hour prior to ischemia (10 mg./kg. orally). Survival, serum urea, serum creatinine, urine volume and creatinine clearance were used to test the effectiveness of the drug. Nisoldipine treatment resulted in the survival of all animals (compared to 66.6 per cent in the untreated group) and improved immediate and long term (14 days) renal function. The drug given post ischemia only was not effective, suggesting that nisoldipine must be present in the kidney during ischemia. The beneficial effects of the drug in postischemic acute renal failure may be attributed in part to effects on postischemic renal hemodynamics. Additional direct effects on ischemic renal epithelial cells, presumably by inhibiting transmembrane calcium fluxes, cannot be excluded. Without an effective protective therapy, reperfusion of the kidney after prolonged periods of normothermic ischemia causes extensive and often irreversible damage. Although the nature of this damage has been well documented,1 its precise cause remains obscure. 2 Recently, considerable interest has been focused on the role of calcium as a possible mediator of irreversible cell damage following a period of ischemia. Anoxic-ischemic injury of cell membranes is associated with a precipitous influx of calcium from the extracellular to the intracellular compartment, and, as a consequence, with the activation of calcium-dependent catabolic processes. 3 An analysis of this process has led to the suggestion that calcium channel blockers may prevent metabolic disturbances and promote functional and structural recovery after ischemia or anoxia. 4 Several experimental studies on myocardial, 4 • 5 hepatic 6 and cerebraF ischemia support this conjecture. Recent results8 • 9 suggest that impaired mitochondrial oxygen uptake and increased cytosolic calcium concentration may be important events also in the pathogenesis of postischemic and toxic acute renal failure (ARF). Recent papers contain conflicting reports regarding the effects of the calcium channel blocker verapamil on postischemic ARF. Some authors observed beneficial effects of the drug in norepinephrine-induced ARF 10 • 11 as well as in clamping models of ARF 12- 14 and others observed no effects in clamping models of postischemic ARF .11 • 15 Although the calcium channel blocking drugs share the ability to competitively inhibit transmembrane calcium fluxes in smooth and cardiac muscle, 16 it has been noted that they are structurally dissimilar, possessing diverse pharmacological properties. Nisoldipine, a new dihydropyridine derivative (structure formula in fig. 1) chemically similar to nifedipine, exerts potent relaxant effects on vascular smooth muscle. Experimentally, nisoldipine has demonstrated favorable effects on ischemic myocardium in the intact animal. 17 The drug also has an antihypertensive effect in various models of experimental hypertension. 17 The present study was undertaken to investigate whether nisoldipine could protect the rat kidney against ischemic damage. A further aim of the study was to evaluate if nisoldipine Accepted for publication July 10, 1985. * Requests for reprints: Urologische Klinik, Ruhr-Universitiit Bochum, Klinikum Marienhospital, Widumer Str. 8, D-4690 Herne 1, Federal Republic of Germany.
could provide additional protective effects in hypothermic ischemia, which might be of clinical relevance in renal surgery and transplantation. MATERIALS AND METHODS
All experiments were performed on male Wistar rats weighing 180 to 220 gm. The animals were housed in individual metabolic cages and maintained on tap water and a standard pellet diet (ssniff-Versuchstier-Alleindiat), to which they had free access pre- and postoperatively. Four days prior to the study, the right kidney was removed through a small flank incision under ether anesthesia. For preparation of the left kidney, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, Abbott) 40 mg./ kg. body weight (b.w.) and placed on a heated table that maintained body temperature between 37C and 38C. The kidney was exposed by a flank incision, freed from perirenal fat and left attached only by its pedicle. Temporary renal ischemia was induced by occlusion of the vascular pedicle with a microvascular clamp (Y asargil aneurysm clip, Aesculap, closing force 0.39 to 0.49 N) for 60 min. In sham operated animals, the kidneys were prepared identically except for clamping the vascular pedicle. The flank was left exposed for a full 60 min. In the rats subjected to hypothermic ischemia, frozen 5 per cent dextrose slush was placed around the kidney during occlusion of the renal pedicle. After completion of surgery and recovery from anesthesia the rats were returned to their cages. Experiments were performed on seven groups as described in table 1. In groups II, IV and VII nisoldipine (Bayer AG) was given in the standard diet four days prior to, and during the three or fourteen day observation period after ischemia. The concentration of the drug in the diet was 300 ppm (mg./kg. diet). Additionally, nisoldipine (10 mg./kg. b.w.) was administered one hour before induction of ischemia by gavage. In group V the drug was given by gavage one hour after declamping (10 mg./kg. b.w.) and in the diet (300 ppm) during the three days observation period after ischemia. Groups III and IV were subdivided, 10 animals of each group observed over a period of fourteen days and the others for three days. Groups I and II were observed for fourteen days after ischemia, and animals of groups V to VII for three days. Animals were examined daily and weighed at regular intervals. Serum creatinine and urea were determined just prior to ischemia (day 0) and on days 1 and 3 in all groups and further
1251
1252
HERTLE AND GARTHOFF
third of the animals being totally anuric on the first day after declamping. Most of these animals died two to six days after the ischemic insult, and in all cases deaths were due to uremia. All animals of this group had raised serum urea and creatinine concentrations and a diminished creatinine clearance on the first, third and seventh day after ischemia and the values had not returned to normal 14 days after ischemia. The peak rise in urea and creatinine occurred on day 3 after ischemia. By day 3 the surviving animals excreted large volumes of dilute urine, more than 200 per cent of the preischemic values (day 7: 27.2 ± 1.9 µl./min. and day 14: 20.5 ± 2.9 µ1./min.; no.= 6). All animals treated with nisoldipine prior and subsequent to normothermic ischemia (group IV) survived the insult (significantly different versus group III on day 3, p <0.0005). Rats of
on days 7 and 14 in groups I to IV after ischemia. On the same days as indicated above, 24 hr. urine samples were collected. Urine volume and creatinine levels were determined. Endogenous creatinine clearance was estimated from the total 24 hr. creatinine excretion and from the serum creatinine value. Improved survival was tested using Fisher's exact test. 18 The remaining data from the different groups or subgroups 1 or 3 days after ischemia were subjected to analysis of variance. This procedure was followed by Dunnett's test19 or Scheffe's test19 to identify the sources of differences; in cases where varianceinstability was detected data were subjected to logarithmic transformation. A p-value of less than 0.05 was considered to be significant. Results are expressed as means ± standard error of the means (SEM), if not differently indicated. RESULTS
Control values before ischemia (day 0) for all tested parameters (mean values ± standard deviations; no. = 110) were: serum urea 9.2 ± 1.6 mmol./1., serum creatinine 62 ± 15 µmol./ 1., urine volume 3. 7 ± 1.6 µ1./min. and endogenous creatinine clearance 518.3 ± 175.7 µl./min. Figure 2 and tables 2-4 summarize the renal function changes following sham operation or ischemia in the 7 groups of animals. Sham operation without and with nisoldipine treatment (groups I and II) had no detrimental effects on renal excretory function over a fourteen day observation period. Rats subjected to 60 min. normothermic ischemia without any protection (group III) developed an ARF with about one
mmol/1 7
,/\,
60
40
20
~,
I I
!6
'
9 / ¾010109
0
~~
,l-----JI,,
,'
g
X
Ti...>]!
~
if'10
10
I O
I 3
',
'<,10
g
9
10
----~ 10A # -!12=--/...!:!=o_ ,/•
~
I 7
days after
14 ischemia or sham operation
FIG. 2. Serum urea concentrations in uninephrectomized rats after sham operation or 60 min. of normothermic ischemia: A sham-operated, untreated (group I), b, sham-operated, nisoldipine treated (group II),• ischemia, untreated (subgroup III), 0 ischemia, nisoldipine treated (subgroup IV). Means ± SEM; number of surviving animals given at symbols. Day 0: values before sham operation or ischemia. Raised serum urea levels throughout observation period in ischemia-untreated group with considerable mortality. No mortality and lower urea levels at all days after ischemia in nisoldipine-treated group. For statistical evaluation refer to tables 3 and 4.
FIG. 1. Chemical structure of nisoldipine (Isobutyl methyl 1,4-dihydro-2,6-diethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate). TABLE
Group
1. Characteristics of experimental groups
9
33 (10/23) 28 (10/18)
III IV V
10
VI VII
10 10
14 14
Sham-operated, untreated Sham-operated, nisoldipine treated (prior and subsequent to sham operation) N ormothermic ischemia, untreated Normothermic ischemia, nisoldipine treated (prior and subsequent to ischemia) N ormothermic ischemia, nisoldipine treated (only subsequent to ischemia) Hypothermic ischemia, untreated Hypothermic ischemia, nisoldipine treated (prior and subsequent to ischemia)
10
I II
Recovery Period (Days)
Protocol.
No.
14/3 14/3 3 3
3
Groups III and IV were subdivided, with 10 animals of each group followed 14 days and 23 animals of group III and 18 animals of group IV followed 3 days after ischemia.
TABLE 2.
Serum creatinine and creatinine clearance after sham operation or 60 min. normothermic ischemia followed over 14 days. Number of surviving animals in brackets. Means ± SEM
I II III IV
(no.= (no.= (no.= (no.=
Creatinine Clearance (µ!./min.)
Serum Creatinine (µmol./1.)
Experimental Group
1
Day 10) 9) 10) 10)
57 ± 56 ± 274 ± 170 ±
2 (10) 2 (9) 23 (9) 15 (10)
For statistical evaluation refer to tables 3 and 4.
3 63 54 302 126
± ± ± ±
7 (10) 1 (9) 33 (7) 31 (10)
7 57 61 171 68
± ± ± ±
2 (10) 6 (9) 18 (6) 2 (10)
14 61 ± 66 ± 104 ± 56 ±
2 (10) 5 (9) 13 (6) 1 (10)
1 610 ± 547 ± 49 ± 144 ±
3 26 29 12 17
609 465 111 338
± ± ± ±
7 70 66 19 44
541 ± 425 ± 199 ± 454 ±
14 46 31 43 38
581 477 425 563
± ± ± ±
40 43 92 52
TABLE
3. .Suruiual, serum creatinine (8Cr), serum urea (SUr), urine volume (U Vol) and creatinine clearance (CrCl) on the first day afte:~ sham
operation or ischemia. _Means ± SEJvf Experimental Group
Survival
I II III
100 100 96.9 100 100 100 100
(%)
IV V VJ Vil
SCr (µmol./1.) 57 56 259 196 300 63 53
±
28**
± ± ± ± ±
2'** 11 14"** 16 2"** ± 3ab**
SUr (mmol./1.) 8.6 8.5 45.1 35.2 47.3 12.0 9.6
± ± ± ± ± ± ±
0.2"** 0.3"** 2.0 2.4"* 2.0 0.6"** o.4•b**
UVol (µ!./min.) 4.4 5.2 5.3 11.1 7.7 6.4 8.2
± ± ± ± ± ± ±
0.4 0.6 0.8 1.1"** 1.5 0.7 0.6
CrCl (µ!./min.) 610.4 54 7 .3 49.7 132.1 40.2 594.0 733.4
± ± ± ± ± ± ±
25.6"** 28. 7"** 10.8 17.1'** 7.4 61.0"** 67.4"**
• Significantly different from group III (corresponding subgroups). b Values of group VJ! significantly different from those of group VI. * p <0.05, ** p <0.01.
TABLE 4.
Survival, serum creatinine (SCr), serum urea (SUr), urine volume (U Vol) and creatinine clearance (CrCl) on third day after sham operation or ischemia. Means ± SEM
Experimental Group
Survival (%)
I
100 100 66.6 100 90 100 100
II III IV V VI VII
SCr (µmol./1.) 63 ± 54± 280 ± 201 ± 264 ± 59 ± 56 ±
7"** la** 23 21 "** 50 3"** 2"**
SUr (mmol./1.) 8.6 9.1 63.7 41.6 57.1 12.0 10.4
± ± ± ± ± ± ±
0.3"** 0.4"** 4.4 4.8"** 5.3 0.5"** 0.3"**
UVol (µ!./min.) 3.7 2.9 13.0 13.3 13.5 7.5 9.3
± ± ± ± ± ± ±
0.3"** 0.7"** 1.5 0.2 1.6 1.0"** 1.0'*
CrCl (µ!./min.) 608.6 464.6 122.2 224. 7 161.2 749.8 831.3
± ± ± ± ± ± ±
69.6"** 66.2"** 20.0 28.9"** 40.9 62.4"** 52.6"**
• Significantly different from group III (corresponding subgroups). * p <0.05, p <0.01.
group IV had significantly lower serum urea, serum creatinine, and higher creatinine clearance on the first and third day after ischemia than the untreated rats (group Animals treated with nisoldipine had a significantly higher urine volume on day 1 after ischemia than the untreated group. On days 7 and 14 after ischemia the nisoldipine treated animals had normal serum urea, serum creatinine and creatinine clearance values, whereas these values were still abnormal in the untreated rats, The severity of ARF in rats in which nisoldipine administration started after declamping (group V) was similar to that of the untreated animals, Rats treated with regional hypothermia alone (group VI) had slightly elevated serum urea values and urine volumes on days 1 and 3 after ischemia, The values of serum creatinine and creatinine clearance on days 1 and 3 after ischemia did not differ from those on day 0, Animals treated with hypothermia in combination with nisoldipine (group VII) had significantly lower serum creatinine and urea concentrations on day 1 after ischemia with group VL All animals of groups VI and VII the insult, DISCUSSION
A period of 60 min, normothermic ischemia after contralateral nephrectomy resulted in a severe and sometimes lethal renal Nisoldipine treatment before inducing w,,rnorn,w. as well as continuing treatment in the period thereafter, was found to be highly effective in preventing uremic death and improving immediate and long term days) renal function. However, when the drug was given after ischemia only, it was not effective, suggesting that nisoldipine must be present in the organ during the ischemic period if the injury is to be reduced, Regional hypothermia had a superior protective effect compared with nisoldipine, Treatment with nisoldipine in addition to external cooling provided only a slight benefit compared to hypothermia alone. The small size of rat kidney, however, does not allow a definite statement on a possible superior protective effect in larger kidneys treated with the calcium channel blocker in combination with external cooling. The beneficial effects of calcium channel blockers on ischemically damaged tissues have been attributed to the blockade of deleterious calcium influx. 4 Although the cellular and molecular mechanisms leading to ischemic cell damage have not been
clarified, substantial evidence exists that the influx of calcium a majo:r role in mediating cellular injury, 3 Schanne et al. 20 showed that a range of membrane-active toxins only caused toxic cell death in the presence of normal extracellular calcium activity, When the extracellular calcium activity was reduced to that of normal intracellular activity, the toxins had little effect. The cell membrane separates extracellular millimolar calcium concentrations from intracellular micromolar calcium concentrations. This very large electrochemical gradient is maintained by the passive permeability of the plasma membranes to calcium ions and by active extrusion of calcium. If the membrane is damaged by ischemia or toxins, calcium readily enters and accumulates within the cell and results in the rapid death of the cell. 3 Elevated cytosolic calcium levels may have many detrimental effects on the cell: activation of calciumstimulated ATP-ase processes which will further deplete the cell of already stressed stores of ATP, activation of membranebound phospholipases resulting in alterations in membranebound enzyme and membrane permeability and increased free fatty acids which may act as detergents. 21 The exact mechanisms that initiate or maintain ARF are not yet clear. Numerous studies have indicated an association bein ca,cOc,~,c Of renal hemodynamics and tween major ARF, 2 However, exact role of cnam,re~ in renal blood flow in ARF continues to be controversial, since in experimental ARF renal blood flow may have returned to normal and yet the ARF persists as assessed by a continued marked reduction in glomerular filtration rate. 2 This disparity in the ratio of renal blood flow to glomerular filtration rate has suggested to many workers that other pathogenetic factors, such as tubular fluid back-leak and tubular obstruction, may play more important roles than a reduction in renal blood flow in various forms of ARF. 2 Venkatachalam et al. 1 have provided evidence that cytoplasmic blebs may constitute the substrate for intraluminal obstruction in ARF, The biochemical abnormalities which may initiate cellular death during renal ischemia, thereby providing substrate for tubular obstruction, have not been defined, Burke and Schrier8 showed that in renal ischemia a progressive increase in mitochondrial calcium accumulation occurs during reflow. These authors stated that an increased cytosolic calcium in epithelial '.,HaU,",vC
1254
HERTLE AND GARTHOFF
cells contributes to cell necrosis, and that this cellular debris provides the substrate for tubular obstruction and the maintenance of ARF. 22 Also, the initial ischemic phase of ARF may involve increased cytosolic calcium in the smooth muscle cells of the afferent arterioles, a phenomenon known to be associated with vasoconstriction. 23 Nisoldipine has potent relaxant effects on vascular smooth muscle. The EC 50 of the drug (the concentration eliciting halfmaximum relaxation) in potassium-induced contractions of isolated rabbit aortic strips is 2.4 X 10-9 mol./1. 17 Thus the beneficial effects of the drug in post-ischemic ARF may be attributed in part to effects on post-ischemic renal hemodynamics. However, attempts to overcome ischemic vasoconstriction directly by intra-renal artery administration of vasodilators and indirectly by the acute suppression of the renin-angiotensin system have generally been unsuccessful. 24 Furthermore, in the same experimental setting we found no benefit from the potent vasodilator minoxidil (unpublished data), suggesting that nisoldipine may have additional "calcium antagonistic" effects on tubular cells. These effects may include inhibition of deleterious calcium overload of ischemically damaged renal epithelial cells, although calcium channels have not been demonstrated to exist in renal tubular cells up to now. Although the results obtained in this study should be extrapolated with caution to the clinical situation, there is increasing appreciation of the potential of pharmacological calcium channel blockade in renal ischemia. Recently, it was reported25 that the incidence of ARF after cardiovascular surgery dropped after calcium channel blockers were used pre- and intraoperatively to improve cardiac function. It would seem that these drugs had an unexpected but decidedly beneficial side effect.
8. 9. 10.
11. 12.
13. 14.
15.
16. 17.
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