Renal Failure, 14(3), 327-332 (1992)
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Calcium Channel Blockers: Protective Effects in Ischemic Acute Renal Failure Jack F. M. Wetzels, MD, Thomas J. Burke, PhD, and Robert W. Schrier, MD Department of Medicine University of Colorado School of Medicine Denver, Colorado
ABSTRACT
Studies in heart, liver, and kidney have provided evidence that calcium is an important factor in cell injury. Calcium channel blockers are used with increasing frequency in ischemic and toxic renal failure. In this review the available data on the effects of calcium channel blockers in animal models of ischemic renal failure are presented and possible mechanisms of protective actions are discussed,
INTRODUCTION
several years ago. During experiments performed in our laboratory we observed that infusion of two chemically dissimilar calcium entry blockers greatly attenuated the severity of norepinephrine-induced ARF in dogs (6). In this review we discuss the available evidence for the protective effects of calcium entry blockers in ischemic ARF. Furthermore we review the potential mechanisms by which calcium entry blockers might exert these protective effects.
The observation of Ca2+ deposits in areas of tissue necrosis in the liver led to the suggestion that Ca2+might be a cause of cell injury (1). Subsequent studies in heart and liver have provided evidence that Ca2+is indeed an important factor in the progression of cell injury (2-5). In 1981 Nayler et al. demonstrated that the calcium entry blocker verapamil could prevent tissue Ca2 overload and cell damage in the ischemic and reperfused myocardium (5). This study and similar studies that followed created the notion that this class of drugs could become important tools in the study and treatment of ischemic cell injury. The importance of Ca2+ as a mediator of ischemic acute renal failure (ARF) and the possible therapeutic role of calcium entry blockers emerged from studies conducted +
CALCIUM CHANNEL BLOCKERS AND ISCHEMIC ARF Overview of Experimental Studies Since 1983 a number of studies have been published detailing the effects of calcium entry blockers in ischemic 327
Copyright 0 1992 by Marcel Dekker, Inc.
Wetzels, Burke, and Schrier
328
Table 1 Effects of Calcium Entry Blockers on Ischemic ARF: Preischemic Administration Estimate of GFR'
Calcium enty blocker
Ren Fail Downloaded from informahealthcare.com by University of North Carolina on 11/10/14 For personal use only.
Ref. Species
Model
Type
Route
Dose
Time
7
Rat
Clamp 60'
Ver
IV
B200pg + 2.0 pglkglmin
- 120-
8
Rat Rat Rat
Clamp NE 40' Clamp + UN
Ver Ver Ver
IA IA IV
5 pglkglmin 5 pglkglmin 40 pglkglmin
-20-+120' -20-+ 120' -20-+120'
9
Dog
Clamp 60'
Ver
IV
B 0.5 mglkg
- 10'
10
Rat
Clamp 70'
Ver
IA
10 pglkglmin
- 15- +85 '
11
Dog
Clamp 60'
Ver
IA
5 pglkglmin
6
Dog
NE 40'
Ver
IA
12
Rat
C l a m p 6 0 ' + UN
Ver
13
Rat
Clamp 45'
+ UN
14
Rat
15
Rat
16
Dog
+ UN Clamp 60' + UN Clamp 180' + UN
17
Rat
Clamp 60'
+ UN
Clamp
Follow-up
+ 360 '
20%
50%
-30-0'
3h
-
60%
30%
5 pglkglmin
-30-0'
24 h
+
16%
100%
IA
1 cg
- 10-0'
-
28%
22%
Ver
IV
16 pglkglmin
- 30-
10%
30%
Ver
IV
B 0.1 mglkg
- 15'
48 h
19%
38%
Nis
Po
10 mglkg
-96-+72 h
72 h
8%
24%
Dilt
IV
5 pglkglmin
-72-+ 168 h
168 h
20%
55%
Nit
IV
0.3 mglkg 1 mglkg
- 1 5 - 12%' 24 h
24-168 h
+ 165 '
-15--12%
+
15% 15%
15% 40%
-
20%
20%
Sheep
Clamp
Ver
IA
B 50 pglkg
-5'
72 h
20
Dog
Clamp60'+ UN Idem
Ver Ver Nic Nic
IA
3 x B 0.5 mg 10 mg 3 x B 0.05 mg 1 mg
-30, 0, 60' -30- +95 ' -30, 0, 60' -30- 95 '
168 h 168 h 168 h 168 h
Ben Nit Nit Nif
N N N
B B B B
-5' -5' -5' -5'
24-96 24-96 24-96 24-96
Fel
IV
B 10 nmol/kg
- 10'
120'
S312d Ver Flun
IV IV IV
B 0.1 mglkg B 1 mglkg B 1 mglkg
-3' -3' -3'
24 h 24 h 24 h
Rat
Clamp 45'
+ UN
Iv
30 pglkg 30 pglkg 300 pglkg 300 pglkg
+
-
' 24 h
19
N IA N
+ + + +
24 h
48 h
23
4% 52% 6%
24 h
-5'
Hemorrhagic shock 150'
4% 1% 12%
100%
B 37.5 pglkg
Dog
15%
50%
IV
22
-
2%
+ +
Flun
+ UN
+
2h
clamp 45 '
Clamp 60'
-
3h 3h 48 h
Rat
Rat
+
6h
18
21
Efficacy Control Treated
h h h h
+ + + + + + +
Ratio 1:2.5 10% 10% 10% 10%
35% 25% 45% 20%
-
20% 20% 20% 20%
50% 20% 50% 20%
+
5%
60%
15% 15% 15%
50% 50% 15%
+ + -
Abbreviations: ARF, acute renal failure; NE, norepinephrinemodel; UN, unilateral nephrectomy; Ver, verapamil; Nis, nisoldipine; Dilt, diltiazem; Nit, nitrendipine; Flun, flunarizine; Nic, nicardipine; Ben, Benidipine; Fel, felodipine; S3 126, dihydrothiempyridine derivative; N ,intravenous, IA, intraarterial; B, bolus. 'GFR, glomerular filtration rate estimated from the available data, either inulin clearance, creatinine clearance, or serum creatinine and BUN levels.
Calcium Channel Blockers
329 Table 2
Effects of Calcium Entry Blockers on Ischemic ARF: Postischemic Administration Calcium entry blocker Ref. Species
Type
Route
Dose
Time
Follow-up
Efficacy Control Treated
NE 40'
Ver
IA
5 pglkglmin
120'
3h
-
1%
1%
10
Clamp 70' + UN
Ver
IA
10 pglkglmin
15'
24 h
-
20%
20%
11
Clamp 60'
Ver
IA
5 pglkglmin
50'
3h
20%
50%
NE
Ver Nif
IA IA
5 pglkglmin 2 pglkglmin
120' 120'
24 h 24 h
+ + +
16% 16%
50% 50%
Nis
PO
10 mglkg
72 h
72 h
-
8%
7%
16
+ UN Clamp 180' + UN
Dilt
IV
5 pglkglmin
168 h
168 h
-
25 %
30%
17
clamp 60'
Nit
IV
B 1 mglkg
24 h
-
15%
15%
18
Clamp 45' + UN
Ver Flun Nif
IV IV IV
990 pglkg
375 pglkg 195 pglkg
0-15' 0-15' 15'
48 h 48 h 48 h
-
15% 20% 20%
20% 20% 20%
2h
+
5%
60%
24 h
-
15%
15%
8
6 Ren Fail Downloaded from informahealthcare.com by University of North Carolina on 11/10/14 For personal use only.
Model
Estimate of GFRU
15
Clamp
22
Hemorrhagic shock 150'
Fel
IV
B 10 nmollkg
- 10'
23
Clamp 45' + UN
S312d
IV
B 0.5 mg/kg
+1'
*
-
Abbreviations: See Table 1.
'See Table I .
ARF (6-23) (Tables 1 and 2). Although it is clearly evident that these studies are quite heterogeneous with respect to the animal species studied, the model of ischemia (norepinephrine, clamping of the renal vasculature, hemorrhagic shock), the timing, type, dose, and route of administration of the calcium entry blockers, and the time and parameters used to evaluate protection, several conclusions can be drawn. Table 1 summarizes those studies in which the calcium entry blocker was administered before the ischemic event. Most studies confirm the protective efficacy of calcium entry blockers against ischemic ARF. Intrarenal administration is more effective than systemic administration (20), and when the drug is given intravenously, the dosage more often than not has a systemic effect (i.e., lowering of blood pressure). When pronounced, this systemic response may attenuate an in vivo protective effect (1 1). Moreover, experiments have been performed using a low drug dose to avoid systemic effects, an approach which may also attenuate protection (8, 12). Verapamil is the drug used most ofen, but other types of calcium entry blockers probably are equally or
even more effective (dihydropyridines, diltiazem). Flunarizine may be an exception to this generalization since in two studies using this drug, no protective effect was observed (18, 23). Table 2 summarizes the studies in which the calcium entry blocker was administered after the ischemic period. In these studies in general no protection is observed. The studies in which a protective effect was demonstrated were all conducted in dogs (6, 11, 22). In these experiments the administration of the calcium entry blocker was immediately followed by a diuresis.
Vascular Component After an ischemic insult to the kidney, profound vasoconstriction can be observed, as evidenced by an increased afferent and efferent arteriolar resistance (24). Data from micropuncture studies also have demonstrated a decrease in the glomerular ultrafiltration coefficient, probably a consequence of msangial contraction (24,25). These increases in contractility of vascular smooth muscle cells
Wetzels, Burke, and Schrier
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330
and mesangial cells perhaps can be explained by an increased Ca2+ influx induced by anoxia (26), or they might have been induced by an increased activity of vasoconstrictors. Vasoconstrictors such as angiotensin II, norepinephrine, and vasopressin are frequently increased in ischemic ARF. Furthermore, anoxia generates the release of vasoconstrictor substances from the endothelium and recently encbthelin, a very potent vasoconstrictor, has emerged as an important contributor to postischemic vasoconstriction (24, 27). Calcium entry blockers might interfere with the spontaneous Ca2+ influx and can prevent vasoconstriction and mesangial contraction induced by the previously mentioned vasoconstrictors (28, 29). A second vascular phenomenon frequently observed after renal ischemia is loss of autoregulation (30, 31). In the norepinephrine models of ARF lowering of blood pressure even gives rise to a paradoxical renal vasoconstriction (32). In the latter model also a hypersensitivity to renal nerve stimulation is observed (32). Calcium entry blockers are able to prevent this paradoxical vasoconstriction and normalize the response to renal nerve stimulation (32). It is unclear whether calcium entry blockers restore autoregulatory capacity in the clamping models of ARF.
Diuretic and Natriuretic Response One of the remarkable findings observed during the use of calcium entry blockers in the treatment of hypertensive patients was the lack of sodium retention (33). In this respect the calcium entry blockers clearly differ from traditional renal vasodilators such as minoxidil and hydralazine, which all cause sodium retention (34). Subsequent studies have clearly demonstrated that calcium entry blockers have diuretic and natriuretic properties. These effects occur within 10 min after intrarenal infusion of the drugs (34). Although the mechanisms of these effects are debated, studies in humans have provided evidence that calcium entry blockers cause natriuresis independent of changes in renal hemodynamics, and that the main site of action is in the proximal tubule (35). In the discussions on the possible mechanisms by which calcium entry blockers protect against ischemic ARF, the role of the solute diuresis has received little attention. However, in the maintenance phase of ischemic ARF, tubular obstruction is important (36). Diuretics such as furosemide and mannitol afford protection against ARF, probably by increasing intratubular pressure, thus opposing the occurrence of tubular obstruction secondary to debris (37,38). The protective efficacy of such drugs is correlated with the increase in electrolyte excretion (38). Therefore, part
of the protective efficacy of calcium entry blockers may relate to the capability of these drugs to increase water and electrolyte excretion. The fact that pretreatment is much more efficacious supports such a conclusion. Also, the lack of in viw efficacy of flunarizine against ischemic ARF may relate to this effect, since flunarizine does not cause a diuresis (39). Furthermore, in the same experimental model in which calcium entry blockers, administered after the ischemic event, were effective, a similar protective effect has been noted with the use of furosemide (37).
Epithelial Cell Component Techniques employing isolated renal proximal tubules or cultured cells have allowed the study of effects of calcium channel blockers on epithelial cell injury independent of its vascular or diuretic effects. Studies in isolated rabbit proximal tubules demonstrated that verapamil substantially improved tubule K+ and adenosine triphosphate (ATP) during recovery from ischemia, and prevented Ca2+ overload (40). We have extended these observations in isolated rat proximal tubules. Verapamil reduced cell injury as assessed by lactate dehydrogenase (LDH) release, when the tubules were subjected to hypoxia or anoxia (41). To confirm that this protective effect was due to calcium channel blockade, and not merely due to a nonspecific action of verapamil, similar results were obtained using the structurally different calcium entry blocker flunarizine (41). Of note, the protective effect of both calcium entry blockers was not observed when hypoxia is prolonged beyond 15 min. In our laboratory we have also used cell culture techniques to further determine the role of Ca2+ in cell damage. Cultured segments of rabbit tubule were subjected to anoxia for 45 min and subsequently reoxygenated. Lowering extracellular Caz in the first 2 h of reoxygenation enhanced cell viability (42). In this cell culture system we subsequently have shown that calcium entry blockers also increase cell viability after anoxia and reoxygenation (43). In our studies on the effects of calcium channel blockers on hypoxic and anoxic tubular injury we have attempted to correlate cell damage and increased C$+ influx. Ca2+ influx was determined using the isotope 45Ca. Because kinetic analyses of Ca2+ uptake curves require a steady state, a situation which does not exist in hypoxic or anoxic tubules, we limited the measurements to the uptake of Ca2+ within 60 sec after adding the isotope. It is generally assumed that this initial phase represents both Ca2+ binding to the cell surface and unidirectional Ca2+ influx into the cell (44).Using this method we were able to +
Calcium Channel Blockers
33 1
demonstrate consistently elevated Ca2 uptake rates in the initial phase of hypoxic and anoxic cell injury. Calcium entry blockers decreased Ca2+ uptake and cell injury (41). These observations suggested that Ca2+ influx during hypoxia might occur via voltage operated channels. It is known that electrolyte gradients for K+ and Na+ and the ensuing membrane potential are important in regulating voltage operated channels in excitable tissues. In hypoxic and anoxic epithelial cells, substantial K+ loss occurs causing membrane depolarization. This led us to address the question whether membrane depolarization might be involved in the opening of voltage operated channels in renal proximal tubules. We have exposed normoxic renal proximal tubules to various drugs (valinomycin, ouabain, amphotericin) or high K+ medium, circumstances known to alter membranepotential (45). These maneuvers all increased Ca2+ uptake, an effect which could be blocked by verapamil. Cell injury as assessed by LDH release occurred only in the valinomycin-treated group, the only group in which tubule ATP was reduced. Thus, as in hypoxic and anoxic tubules, 4sCa2+ uptake correlated with cell injury only in those circumstances in which ATP levels were reduced. However, it is clear that at least one component of the protective effect of verapamil in hypoxic or ischemic injury is related to its propensity to alter Ca2+ uptake in cells in which the transmembrane K gradient has decreased.
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+
+
CONCLUSION Experimental evidence suggests that Ca2+ is important in cell injury. Animal studies have demonstrated that pretreatment with calcium entry blockers ameliorates ischemic ARF. The exact mechanism of protection is as yet unknown. Controlled studies in humans are warranted, to further assess the clinical relevance of such a treatment modality. Correspondenceand reprint requests to: Dr. Robat W. Schrier, C281, University of Colorado School of Medicine, 4200 E. 9th Ave., Denver, CO 80262. This work was supported by a grant from the National Institutes of Health (DK 35098). Dr. Wetzels was supported by a grant from the Dutch Kidney Foundation (Nierstichting Nederknd-C90.0958). The authors would like to thank Linda M. Benson for secretarial assistance.
REFERENCES 1. McLean AEM, McLean E, Judah JD: Cellular necrosis in the liver induced and modified by drugs Inr RevErp Path01 4: 127-157, 1%5.
2. Shen AC, Jennings RB: Myocardial calcium and magnesium in acute ischemic injury. Am J Parhol67:417-440, 1972. 3. Schanne FAX, Kane AB, Young EE, Farber JL: Calcium dependence of toxic cell death: a final common pathway. Science 206:700-702, 1979. Farber JL: The role of calcium in cell death. Life Sci 29: 1289-1295, 1981. Nayler WG: The role of calcium in the ischemic myocardium. Am J Path01 102:262-270, 1981. Burke TJ, Amold PE, Gordon JA, Bulger RE, h b y a n DC,Schrier RW: Protective effect of intrarenal calcium membrane blockers before and after renal ischemia. Functional, morphological, and mitochondria1 studies. J Clin Invest 74: 1830-1841, 1984. 7. Kramer HJ, Neumark A, Schmidt S , Klingmuller D, Glanzer K: Renal functional and metabolic studies in the role of preventive measures in experimental acute ischemic renal failure. Clin &p Dialysis Apheresis 7:77-99, 1983. 8. Malis CD, Cheung JY, Leaf A, Bonventre JV:Effects of verapamil in models of ischemic acute renal failure in the rat. Am J Physiol 245:F735-F742, 1983. 9. Papadimitriou M, Alexopoulos E, Vargemezis V, Sakellariou G, Kosmidou I, Metaxas P: The effect of preventive administration of verapamil on ischaemic renal failure in dogs. Proc EDTA 20:650-655, 1983. ~ Serban I, Gavendo S , Kapuler S , Eliahou 10. Goldfarb D, I a i A, HE: Beneficialeffect of verapamil in ischemic acute renal failure in the rat. Prm Soc Exp Biol Med 172:389-392, 1983. 11. Wait RB, White G,Davis JH: Beneficial effects of verapamil on postischemic renal failure. Surgery 94:276282, 1983. 12. Blank W, Unni Mooppan MM, Chhajwanni B, Chou SY, Kim H: Effects of verapamil on preservation on renal function after ischemia: Functional and ultrastructuralstudy. J Urol13 1 :992-994, 1984. 13. Ishigami M, Magnusson MO, Stowe NT, Straffon RA: The salutary effect of verapamil and d-propanolol in ischemically damaged kidneys. Transplant Pmc 16:40-43, 1984. 14. Gingrich GA, Barker GR, Lui P, Stewart SC: Renal preservation following severe ischemia and prophylactic calcium channel blockade. J Uml 134:408-410, 1985. 15. Hertle L, Garthoff B: Calcium channel blocker nisoldipine limits ischemic damage in rat kidney. J Urol 134:1251-1254, 1985. 16. Wagner K.Schultze G,Molzahn M, Neumayer HH: The influence of long-term infusion of the calcium antagonist diltiazem on postischemic a a t e renal failure in corsciousdogs. Klin Wochnschr 64:135-140, 1986. 17. Rose H, Philipson J, Puschen JB: Effect of nitrendipine in a rat model of ischemic acute renal failure. J Cardiovasc Pharmacol ~ ( S U P 1):S57-S59, P~ 1987. 18. Leahy AL, Fitazpatrick JM, Waite RB: Variable results of calcium blockade in post-ischemic renal failure. Eur Urol 14:222-225, 1988. 19. Woolley JL, Barker GR, Jacobsen WK, et al: Effect of the calcium entry blocker verapamil on renal ischemia. Cn't Care Med 16148-51, 1988. 20. Elkadi HK, Mardan AH, Nghiem DD, Southard JH: The role of calcium antagonists in the management of renal warm ischemia. J Urol 141:974-980, 1989. 21. Karasawa A, Kubo K: Protection by benidipine hydrochloride (KW-3049), a calcium antagonist, of ischemic kidney in rats via inhibitions of Ca2+overload, ATP decline and lipid peroxidation. Japan J Pharmacol 52553-562, 1990.
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332 22. Chintala MS, Jandhyala BS: Renal failure in haemorrhagic shock in dogs: salutary effecb of the calcium entry blocker felodipine. Naunyn-Schmiedeberg S Arch Pharmacol 341 :357-363, 1990. 23. Shimizu T. Kawabata T, Nakamura M: Protective effect of a novel calcium blocker, S-312-d, on ischemic acute renal failure in rat. J Pharmacol Exp n e r 255:484-490, 1990. 24. Kon V, Yoshioka T, Fogo A, Ichikawa I: Glomerular actions of endothelin in M'vo. J Clin Invesr 83:1762-1767, 1989. 25. Williams RH, lkomas CE, Navar LG,Evan AP: Hemodynamic and single nephron function during the maintenance phase of ischemic acute renal failure in the dog. Kidney Int 19:M3-515, 1981. 26. Vanhoutte PM: Effects of anoxia and glucose depletion on isolated veins of the dog. Am J Physiol2301261-1268, 1971. 27. Rubanyi GM, Vanhoutte PM: Hypoxia releases a vasoconstrictor substance from the canine vascular endahelium. J Physiol 364145-56, 1985. 28. Ichikawa I, Miele JF, Brenner BM: Reversal of renal cortical actions of angiotensin I1 by verapamil and manganese. Kidney Int 16:137-147, 1979. 29. Goldberg JD, Schrier RW: Effect of calcium membrane blockers on in vivo vasoconstrictor properties of norepinephrine, angiotensin II, and vasopressin. Mineral Electrolyte Met& 10:178-183, 1984. 30. Adams PL, Adams FF, Bell PD, Navar LG: Impaired renal blood flow autoregulation in ischemic acute renal failure. Kidney Int 18~68-76,1980, 31. Matthys E, Paaon MK, Osgood RW, Venkatachalam MA, Stein JH: Alterations in vascular function and morphology in acute ischemic renal failure. Kidney Int 23:717-724, 1983. 32. Conger JD, Robinette JB, Schrier RW: Smooth muscle calcium and endotheliumderived relaxing factor in the abnormal vascular responses of a a t e renal failure. J a i n Invest 82532-537, 1988. 33. Zanchetti A, Leonetti G:Natriuretic effects of calcium antagonists. J Cardiovasc Pharmaml 7(suppl 4):S33-S37, 1985. 34. DiBona GF: Effects of vasodilator antihypertensive agents on renal function. J Cardiovasc Pharmacol 9(suppl l):S14-S16, 1987.
Wetzels, Burke, and Schrier 35. Wetzels JFM, Wiltink PG, Hoitsmakl, Huysmans FThM, Koene RAP: Diuretic and natriuretic effects of nifedipine in healthy persons. Br J Clin Pharmacol25:547-553. 1988. 36. Tanner GA, Scphosan S:Kidney pressures after temporary renal artery occlusion in the rat. Am JPhysiol230:1173-1181, 1975. 37. de Torrente A, Miller PD, Cronin RE, Paulsen PE, Erickson AL, Schrier RW: Effects of furosemide and acetylcholine in norepinephrine-induced acute renal failure.Am JPhysiol235:F131-F135, 1978. 38. Cronin RE, de Torrente A, Miller PD, Bulger RE, Burke TJ, Schrier RW: Pathogenic mechanisms in early norepinephrineinduced acute renal failure: Functional and histological correlates of protection. Kidney Int 14:115-125, 1978. 39. Vemulapalli S, Chiu PJ, Sybertz EJ: Comparative renal effects of calcium channel blockers in conscious spontaneously hypertensive rats. Arch Int Ph-codyn Ther 287:30!-322, 1987. 40. Humes HD, Hunt DA, Clark MJ, White MP. Weinberg JM: Cellular mechanisms of protection in nephrotoxic and ischemic acute renal failure. In Robinson RR (ed), Nephrology, Vol. I , New York, Springer-Verlag, 1984, pp. 776-783. 41. Almeida ARP, Bunnachak D, Burnier M, Wetzels JFM, Burke TJ, Schrier RW Time dependent protective effects of calcium channel blockers on anoxia and hypoxia induced proximal tubular injury. J P h a m o l Erp n e r 260326432, 1992. 42. Wilson PD. Schrier RW: Nephron segment and calcium as determinants of anoxic cell death in primary renal cell cultures. Kidney Int 29: 1172-1 179, 1986. 43. Schwertschlag U , Schrier RW, Wilson P: Beneficial effects of calcium channel blockers and calmodulin binding drugs on in virro renal cell anoxia. J Pharmacol Exp n e r 238:119-124, 1986. 44. Snowdowne KW, Freudenrich CC, Borle AB:The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells. J Biol Chem 260:11619-11626, 1985. 45. Schrier RW, Conger JD, Burke TJ: Pathogenetic role of calcium in renal cell injury. In Hatano M (ed), Nephrology. Tokyo, Springer-Verlag. 1991, pp. 648-659.
Ren Fail Downloaded from informahealthcare.com by University of North Carolina on 11/10/14 For personal use only.
Calcium Channel Blockers: Protective Effects in Ischemic Acute Renal Failure Jack F. M. Wetzels, MD, Thomas J. Burke, PhD, and Robert W. Schrier, MD Department of Medicine University of Colorado School of Medicine Denver, Colorado
ABSTRACT
Studies in heart, liver, and kidney have provided evidence that calcium is an important factor in cell injury. Calcium channel blockers are used with increasing frequency in ischemic and toxic renal failure. In this review the available data on the effects of calcium channel blockers in animal models of ischemic renal failure are presented and possible mechanisms of protective actions are discussed,
INTRODUCTION
several years ago. During experiments performed in our laboratory we observed that infusion of two chemically dissimilar calcium entry blockers greatly attenuated the severity of norepinephrine-induced ARF in dogs (6). In this review we discuss the available evidence for the protective effects of calcium entry blockers in ischemic ARF. Furthermore we review the potential mechanisms by which calcium entry blockers might exert these protective effects.
The observation of Ca2+ deposits in areas of tissue necrosis in the liver led to the suggestion that Ca2+might be a cause of cell injury (1). Subsequent studies in heart and liver have provided evidence that Ca2+is indeed an important factor in the progression of cell injury (2-5). In 1981 Nayler et al. demonstrated that the calcium entry blocker verapamil could prevent tissue Ca2 overload and cell damage in the ischemic and reperfused myocardium (5). This study and similar studies that followed created the notion that this class of drugs could become important tools in the study and treatment of ischemic cell injury. The importance of Ca2+ as a mediator of ischemic acute renal failure (ARF) and the possible therapeutic role of calcium entry blockers emerged from studies conducted +
CALCIUM CHANNEL BLOCKERS AND ISCHEMIC ARF Overview of Experimental Studies Since 1983 a number of studies have been published detailing the effects of calcium entry blockers in ischemic 327
Copyright 0 1992 by Marcel Dekker, Inc.
Wetzels, Burke, and Schrier
328
Table 1 Effects of Calcium Entry Blockers on Ischemic ARF: Preischemic Administration Estimate of GFR'
Calcium enty blocker
Ren Fail Downloaded from informahealthcare.com by University of North Carolina on 11/10/14 For personal use only.
Ref. Species
Model
Type
Route
Dose
Time
7
Rat
Clamp 60'
Ver
IV
B200pg + 2.0 pglkglmin
- 120-
8
Rat Rat Rat
Clamp NE 40' Clamp + UN
Ver Ver Ver
IA IA IV
5 pglkglmin 5 pglkglmin 40 pglkglmin
-20-+120' -20-+ 120' -20-+120'
9
Dog
Clamp 60'
Ver
IV
B 0.5 mglkg
- 10'
10
Rat
Clamp 70'
Ver
IA
10 pglkglmin
- 15- +85 '
11
Dog
Clamp 60'
Ver
IA
5 pglkglmin
6
Dog
NE 40'
Ver
IA
12
Rat
C l a m p 6 0 ' + UN
Ver
13
Rat
Clamp 45'
+ UN
14
Rat
15
Rat
16
Dog
+ UN Clamp 60' + UN Clamp 180' + UN
17
Rat
Clamp 60'
+ UN
Clamp
Follow-up
+ 360 '
20%
50%
-30-0'
3h
-
60%
30%
5 pglkglmin
-30-0'
24 h
+
16%
100%
IA
1 cg
- 10-0'
-
28%
22%
Ver
IV
16 pglkglmin
- 30-
10%
30%
Ver
IV
B 0.1 mglkg
- 15'
48 h
19%
38%
Nis
Po
10 mglkg
-96-+72 h
72 h
8%
24%
Dilt
IV
5 pglkglmin
-72-+ 168 h
168 h
20%
55%
Nit
IV
0.3 mglkg 1 mglkg
- 1 5 - 12%' 24 h
24-168 h
+ 165 '
-15--12%
+
15% 15%
15% 40%
-
20%
20%
Sheep
Clamp
Ver
IA
B 50 pglkg
-5'
72 h
20
Dog
Clamp60'+ UN Idem
Ver Ver Nic Nic
IA
3 x B 0.5 mg 10 mg 3 x B 0.05 mg 1 mg
-30, 0, 60' -30- +95 ' -30, 0, 60' -30- 95 '
168 h 168 h 168 h 168 h
Ben Nit Nit Nif
N N N
B B B B
-5' -5' -5' -5'
24-96 24-96 24-96 24-96
Fel
IV
B 10 nmol/kg
- 10'
120'
S312d Ver Flun
IV IV IV
B 0.1 mglkg B 1 mglkg B 1 mglkg
-3' -3' -3'
24 h 24 h 24 h
Rat
Clamp 45'
+ UN
Iv
30 pglkg 30 pglkg 300 pglkg 300 pglkg
+
-
' 24 h
19
N IA N
+ + + +
24 h
48 h
23
4% 52% 6%
24 h
-5'
Hemorrhagic shock 150'
4% 1% 12%
100%
B 37.5 pglkg
Dog
15%
50%
IV
22
-
2%
+ +
Flun
+ UN
+
2h
clamp 45 '
Clamp 60'
-
3h 3h 48 h
Rat
Rat
+
6h
18
21
Efficacy Control Treated
h h h h
+ + + + + + +
Ratio 1:2.5 10% 10% 10% 10%
35% 25% 45% 20%
-
20% 20% 20% 20%
50% 20% 50% 20%
+
5%
60%
15% 15% 15%
50% 50% 15%
+ + -
Abbreviations: ARF, acute renal failure; NE, norepinephrinemodel; UN, unilateral nephrectomy; Ver, verapamil; Nis, nisoldipine; Dilt, diltiazem; Nit, nitrendipine; Flun, flunarizine; Nic, nicardipine; Ben, Benidipine; Fel, felodipine; S3 126, dihydrothiempyridine derivative; N ,intravenous, IA, intraarterial; B, bolus. 'GFR, glomerular filtration rate estimated from the available data, either inulin clearance, creatinine clearance, or serum creatinine and BUN levels.
Calcium Channel Blockers
329 Table 2
Effects of Calcium Entry Blockers on Ischemic ARF: Postischemic Administration Calcium entry blocker Ref. Species
Type
Route
Dose
Time
Follow-up
Efficacy Control Treated
NE 40'
Ver
IA
5 pglkglmin
120'
3h
-
1%
1%
10
Clamp 70' + UN
Ver
IA
10 pglkglmin
15'
24 h
-
20%
20%
11
Clamp 60'
Ver
IA
5 pglkglmin
50'
3h
20%
50%
NE
Ver Nif
IA IA
5 pglkglmin 2 pglkglmin
120' 120'
24 h 24 h
+ + +
16% 16%
50% 50%
Nis
PO
10 mglkg
72 h
72 h
-
8%
7%
16
+ UN Clamp 180' + UN
Dilt
IV
5 pglkglmin
168 h
168 h
-
25 %
30%
17
clamp 60'
Nit
IV
B 1 mglkg
24 h
-
15%
15%
18
Clamp 45' + UN
Ver Flun Nif
IV IV IV
990 pglkg
375 pglkg 195 pglkg
0-15' 0-15' 15'
48 h 48 h 48 h
-
15% 20% 20%
20% 20% 20%
2h
+
5%
60%
24 h
-
15%
15%
8
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Model
Estimate of GFRU
15
Clamp
22
Hemorrhagic shock 150'
Fel
IV
B 10 nmollkg
- 10'
23
Clamp 45' + UN
S312d
IV
B 0.5 mg/kg
+1'
*
-
Abbreviations: See Table 1.
'See Table I .
ARF (6-23) (Tables 1 and 2). Although it is clearly evident that these studies are quite heterogeneous with respect to the animal species studied, the model of ischemia (norepinephrine, clamping of the renal vasculature, hemorrhagic shock), the timing, type, dose, and route of administration of the calcium entry blockers, and the time and parameters used to evaluate protection, several conclusions can be drawn. Table 1 summarizes those studies in which the calcium entry blocker was administered before the ischemic event. Most studies confirm the protective efficacy of calcium entry blockers against ischemic ARF. Intrarenal administration is more effective than systemic administration (20), and when the drug is given intravenously, the dosage more often than not has a systemic effect (i.e., lowering of blood pressure). When pronounced, this systemic response may attenuate an in vivo protective effect (1 1). Moreover, experiments have been performed using a low drug dose to avoid systemic effects, an approach which may also attenuate protection (8, 12). Verapamil is the drug used most ofen, but other types of calcium entry blockers probably are equally or
even more effective (dihydropyridines, diltiazem). Flunarizine may be an exception to this generalization since in two studies using this drug, no protective effect was observed (18, 23). Table 2 summarizes the studies in which the calcium entry blocker was administered after the ischemic period. In these studies in general no protection is observed. The studies in which a protective effect was demonstrated were all conducted in dogs (6, 11, 22). In these experiments the administration of the calcium entry blocker was immediately followed by a diuresis.
Vascular Component After an ischemic insult to the kidney, profound vasoconstriction can be observed, as evidenced by an increased afferent and efferent arteriolar resistance (24). Data from micropuncture studies also have demonstrated a decrease in the glomerular ultrafiltration coefficient, probably a consequence of msangial contraction (24,25). These increases in contractility of vascular smooth muscle cells
Wetzels, Burke, and Schrier
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330
and mesangial cells perhaps can be explained by an increased Ca2+ influx induced by anoxia (26), or they might have been induced by an increased activity of vasoconstrictors. Vasoconstrictors such as angiotensin II, norepinephrine, and vasopressin are frequently increased in ischemic ARF. Furthermore, anoxia generates the release of vasoconstrictor substances from the endothelium and recently encbthelin, a very potent vasoconstrictor, has emerged as an important contributor to postischemic vasoconstriction (24, 27). Calcium entry blockers might interfere with the spontaneous Ca2+ influx and can prevent vasoconstriction and mesangial contraction induced by the previously mentioned vasoconstrictors (28, 29). A second vascular phenomenon frequently observed after renal ischemia is loss of autoregulation (30, 31). In the norepinephrine models of ARF lowering of blood pressure even gives rise to a paradoxical renal vasoconstriction (32). In the latter model also a hypersensitivity to renal nerve stimulation is observed (32). Calcium entry blockers are able to prevent this paradoxical vasoconstriction and normalize the response to renal nerve stimulation (32). It is unclear whether calcium entry blockers restore autoregulatory capacity in the clamping models of ARF.
Diuretic and Natriuretic Response One of the remarkable findings observed during the use of calcium entry blockers in the treatment of hypertensive patients was the lack of sodium retention (33). In this respect the calcium entry blockers clearly differ from traditional renal vasodilators such as minoxidil and hydralazine, which all cause sodium retention (34). Subsequent studies have clearly demonstrated that calcium entry blockers have diuretic and natriuretic properties. These effects occur within 10 min after intrarenal infusion of the drugs (34). Although the mechanisms of these effects are debated, studies in humans have provided evidence that calcium entry blockers cause natriuresis independent of changes in renal hemodynamics, and that the main site of action is in the proximal tubule (35). In the discussions on the possible mechanisms by which calcium entry blockers protect against ischemic ARF, the role of the solute diuresis has received little attention. However, in the maintenance phase of ischemic ARF, tubular obstruction is important (36). Diuretics such as furosemide and mannitol afford protection against ARF, probably by increasing intratubular pressure, thus opposing the occurrence of tubular obstruction secondary to debris (37,38). The protective efficacy of such drugs is correlated with the increase in electrolyte excretion (38). Therefore, part
of the protective efficacy of calcium entry blockers may relate to the capability of these drugs to increase water and electrolyte excretion. The fact that pretreatment is much more efficacious supports such a conclusion. Also, the lack of in viw efficacy of flunarizine against ischemic ARF may relate to this effect, since flunarizine does not cause a diuresis (39). Furthermore, in the same experimental model in which calcium entry blockers, administered after the ischemic event, were effective, a similar protective effect has been noted with the use of furosemide (37).
Epithelial Cell Component Techniques employing isolated renal proximal tubules or cultured cells have allowed the study of effects of calcium channel blockers on epithelial cell injury independent of its vascular or diuretic effects. Studies in isolated rabbit proximal tubules demonstrated that verapamil substantially improved tubule K+ and adenosine triphosphate (ATP) during recovery from ischemia, and prevented Ca2+ overload (40). We have extended these observations in isolated rat proximal tubules. Verapamil reduced cell injury as assessed by lactate dehydrogenase (LDH) release, when the tubules were subjected to hypoxia or anoxia (41). To confirm that this protective effect was due to calcium channel blockade, and not merely due to a nonspecific action of verapamil, similar results were obtained using the structurally different calcium entry blocker flunarizine (41). Of note, the protective effect of both calcium entry blockers was not observed when hypoxia is prolonged beyond 15 min. In our laboratory we have also used cell culture techniques to further determine the role of Ca2+ in cell damage. Cultured segments of rabbit tubule were subjected to anoxia for 45 min and subsequently reoxygenated. Lowering extracellular Caz in the first 2 h of reoxygenation enhanced cell viability (42). In this cell culture system we subsequently have shown that calcium entry blockers also increase cell viability after anoxia and reoxygenation (43). In our studies on the effects of calcium channel blockers on hypoxic and anoxic tubular injury we have attempted to correlate cell damage and increased C$+ influx. Ca2+ influx was determined using the isotope 45Ca. Because kinetic analyses of Ca2+ uptake curves require a steady state, a situation which does not exist in hypoxic or anoxic tubules, we limited the measurements to the uptake of Ca2+ within 60 sec after adding the isotope. It is generally assumed that this initial phase represents both Ca2+ binding to the cell surface and unidirectional Ca2+ influx into the cell (44).Using this method we were able to +
Calcium Channel Blockers
33 1
demonstrate consistently elevated Ca2 uptake rates in the initial phase of hypoxic and anoxic cell injury. Calcium entry blockers decreased Ca2+ uptake and cell injury (41). These observations suggested that Ca2+ influx during hypoxia might occur via voltage operated channels. It is known that electrolyte gradients for K+ and Na+ and the ensuing membrane potential are important in regulating voltage operated channels in excitable tissues. In hypoxic and anoxic epithelial cells, substantial K+ loss occurs causing membrane depolarization. This led us to address the question whether membrane depolarization might be involved in the opening of voltage operated channels in renal proximal tubules. We have exposed normoxic renal proximal tubules to various drugs (valinomycin, ouabain, amphotericin) or high K+ medium, circumstances known to alter membranepotential (45). These maneuvers all increased Ca2+ uptake, an effect which could be blocked by verapamil. Cell injury as assessed by LDH release occurred only in the valinomycin-treated group, the only group in which tubule ATP was reduced. Thus, as in hypoxic and anoxic tubules, 4sCa2+ uptake correlated with cell injury only in those circumstances in which ATP levels were reduced. However, it is clear that at least one component of the protective effect of verapamil in hypoxic or ischemic injury is related to its propensity to alter Ca2+ uptake in cells in which the transmembrane K gradient has decreased.
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+
+
CONCLUSION Experimental evidence suggests that Ca2+ is important in cell injury. Animal studies have demonstrated that pretreatment with calcium entry blockers ameliorates ischemic ARF. The exact mechanism of protection is as yet unknown. Controlled studies in humans are warranted, to further assess the clinical relevance of such a treatment modality. Correspondenceand reprint requests to: Dr. Robat W. Schrier, C281, University of Colorado School of Medicine, 4200 E. 9th Ave., Denver, CO 80262. This work was supported by a grant from the National Institutes of Health (DK 35098). Dr. Wetzels was supported by a grant from the Dutch Kidney Foundation (Nierstichting Nederknd-C90.0958). The authors would like to thank Linda M. Benson for secretarial assistance.
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