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Calcium antagonists improve kidney function in the rat after cold storage in high-Na UW but not in high-K UW solution
ELSEVIER
Calcium Antagonists Improve Kidney Function in the Rat After Cold Storage in High-Na UW but Not in High-K UW Solution A. Hadj-ATssa, S.C. Ramella-Virieux, J.P. Steghens, A. Barbieux, and N. Pozet X T R A C E L L U L A R types (high-Na) of cold-storage solution (CSS) have been shown to be more effective in preserving kidneys than intracellular CSS (high-K). 1"2We have previously demonstrated with the isolated perfused rat kidney model (IPK) that a high-Na version of University of Wisconsin (UW) Belzer's CSS (Na-UW) seemed to be less injurious to recovery function than the original high-K UW Belzer's solution (K-UW). 3 On the other hand, a number of clinical investigations have suggested that calcium entry blockers (CEB) may improve graft function when administered after and/or prior to transpiantation. 4"5 The ischemia reperfusion syndrome involves, in part, an alteration in intracellular calcium metabolism that induces an increase in renal vascular resistances (RVR) and other cellular dysfunction, and high-K CSS per se are vasoconstrictive.6 Since maneuvers that would prevent vasoconstriction may favour the intraorgan diffusion of the cold-storage solution and ameliorate preservation, we evaluated with the IPK model, the actual benefit of the vasodilator nifedipine on kidneys preserved in K-UW and Na-UW.
E
MATERIALS AND METHODS The IPK Technique
Sprague-Dawley rats (weighing 200 to 250 g) were used. Unless otherwise indicated, the technique for IPK has been previously described in detail.3 Cold-Storage Solutions
Two CSS, derived from UW Belzer's solution and prepared in the pharmaceutical department,3 were compared: (1) K-UW solution was identical to the original Belzer's high-potassium UW solution (125 mmol/L potassium and 30 mmol/L sodium); and (2) Na-UW solution contained the same agents as the original UW solution but with high-sodium and low-potassium concentrations (125 mmol/L sodium and 30 mmol/L potassium). EXPERIMENTAL PROTOCOLS AND RESULTS
The IPK was used, first as a vascular bed to test the effects of CSS on renal vascular resistances and the influence of nifedipine. Second, the recovery function of the IPK was assessed by glomerular filtration rate (GFR, inulin clearance) and tubular Na reabsorption, after 24-hour preservation in K-UW and Na-UW, with or without nifedipine. Results were compared with a control group in which renal function was measured without prior cold storage. © 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Transplantation Proceedings, 29, 2439-2441 (1997)
Effects of Cold-Storage Solutions on Renal Vascular Resistances of the IPK
The kidneys were placed in the isolated perfusion circuit and perfused with Krebs-Henseleit solution without recirculation of the effluent, at a rate of 10 mL/min until stabilization of renal perfusion pressure. Therefore, the CSS was infused through the main perfusate line during 1 minute. When the CSS infusion was started, the perfusion of Krebs-Henseleit was stopped so that the kidney was constantly perfused at a rate of 10 mL/min. As a result, recorded changes in perfusion pressure are actually in relation to changes in RVR and not in the rate of perfusion. For each cold-storage solution, four rats were used. The infusion of K-UW induced significant increases in perfusion pressure of 90 _+ 20 mm Hg. Conversely, an identical infusion rate of Na-UW did not induce any significant change in perfusion pressure (+9.5 _+ 0.6 mm Hg). Effects of Nifedipine on the Changes in RVR Induced by the CSS
After cannulation of the kidney and its perfusion with Krebs-Henseleit solution as described above, the CSS was infused during 1 minute every 5 minutes for 10 times, at a rate of 10 mL/min. When the CSS was started, the KrebsHenseleit perfusion was stopped. After the CSS had been infused for three consecutive times, to confirm the reproducibility of the induced changes in perfusion pressure, an infusion of nifedipine (10 6 mol/L in NaCI 0.9%) was added during 10 minutes and then withdrawn. For each CSS, four rats were used. Before adding nifedipine, the infusion of K-UW for three consecutive times induced a reproducible increase in perFrom the Department of Renal Functional Exploration (A.H.-A., S.G.R.-V., N.P.), Laboratory of Biochemistry C (J.P.S.), and Pharmaceutical Department (A.B.), Edouard Herriot Hospital and Claude Bernard University, Lyon, France. Supported by the Conseil General du Rh6ne and by grants from the Hospices Civils de Lyon and the Fondation de rAvenir, France. Address reprint requests to Dr A. Hadj-Aissa, Service d'Exploration Fonctionnelle Renale, H6pital Edouard Herriot, Pavilion P, 69437 Lyon Cedex 03, France. 0041-1345/97/$17.00 PII S0041-1345(97)00441-7 2439
2440
HADJ-AiSSA, RAMELLA-VIRIEUX, STEGHENS ET A L
Table 1. Glomemlar Filtration Rate (GFR), Fractional Na Reabsorption (FR.a), Absolute Na Reabsorption (AR..), Perfusion Pressure (PP), and Renal Vascular Resistances (RVR) in Control IPK and in Kidneys After 24 Hours of Cold Storage in K-UW and Na-UW, With (+) or Without (-) addition of Nifedipine Control Nifedipine (-) GFR (/zL/rnin/g) FRN, (%) ARNa (/zmol/min/g) PP (mm Hg) RVR (mm Hg/mL/min/g)
441 91.4 55.48 99.7 5.3
-- 54 -+ 1.7 _+ 6.14 -+ 0.8 -+ 0.7
K-UW Nifedipine (+)
305 86.9 37.90 98.8 5.1
-+ 51 _+ 3.2 _+ 7.34 -+ 1.8 _+ 0.6
Na-UW
Nifedipine (-)
Nifedipine (+)
49 26.7 2.26 97.4 5.3
41 34.4 2.32 98.0 5.8
+ t1" _+ 1.9" _+ 0.69* _+ 0.35 -+ 0.5
_+ 13" _+ 7.4* _+ 1.19" _+ 2.4 _+ 0.6
Nifedipine (-) 52 58.2 4.61 99.7 5.8
_+ 10" _+ 7.4 *t _ 0.97 *t _+ 1.4 _+ 0.5
Nifedipine (+) 129 74.2 13.04 99.2 4.9
+ 21 *it _+ 4.9 *t _+ 2.15 *tt _+ 0.7 _+ 0.4
*P < .05 vs control. tp < .05 vs K-UW. tP < .05 vs nifedipine (-).
fusion pressure of 54 ± 13 mm Hg above base-line. In similar conditions, the infusion of Na-UW induced a slight, not significant, decrease in perfusion pressure of 8 - 3 mm Hg below base-line. The addition of nifedipine to K-UW produced a significant reduction of the peak perfusion pressure to only 17 -+ 6 mm Hg, whereas Na-UW infusion was not influenced by nifedipine ( - 3 -+ 1 mm Hg).
stored with K-UW. In contrast, compared to Na-UW N - , the addition of nifedipine to kidneys stored in Na-UW induced a significant increase in GFR and ARNa. FRNa increased, but not significantly. Table 1 shows that renal function of Na-UW-nifedipine became significantly higher than that of K-UW N+. There was no difference in PP or RVR between K-UW, Na-UW, and control.
Influence o f Nifedipine on Renal F u n c t i o n o f t h e IPK (Table 1)
DISCUSSION
Without Nifedipine (N-). In the control group N - (n = 6), the kidneys were perfused normothermically with the Krebs-Henseleit-albumine solution immediately after they were harvested. Results are shown in Table 1. In the cold-stored group N - , kidneys were catheterized and isolated without interruption of blood flow and flushed with the cold-storage solution cooled at +4°C for 10 minutes at 3 mL/min. The kidneys were then placed with their catheters in a small container with 30 mL of the cold-storage solution and stored at 0-4°C for 24 hours. Afterward, the organs were reperfused on the isolated perfusion circuit with perfusion medium at 37°C to measure renal function and hemodynamics. In these experiments 14 rats were used, 7 for each cold-storage solution. After 24 hours of cold storage in each of the two solutions, GFR was dramatically decreased by 90% compared to control. Na reabsorption was also considerably altered by cold storage. However, both fractional Na reabsorption (FRNa) and absolute Na reabsorption (ARNa) of kidneys preserved in Na-UW were significantly higher than in K-UW. There was no difference in PP or RVR between cold-stored kidneys and control. With Nifedipine (N+). The same experiments were performed as indicated above, but nifedipine 10 - 6 mol/L was added to the cold-storage solution and to the KrebsHenseleit perfusion medium. In these last experiments, 18 rats were used: six controls and six for each cold-storage solution. In the control group N+, the addition of nifedipine did not cause any significant alteration in the functional parameters (Table 1). In the cold-stored group N+, nifedipine did not significantly influence renal function of kidneys flushed and
Cold-storage solutions commonly used for preservation of organs before transplantation such as Euro-Collins (EC) and Belzer's UW solutions have a high potassium concentration. These solutions are supposed to limit potassium leakage from stored cells and thereby maintain a more normal intracellular milieu. However, high potassium-containing solutions have several disadvantages. In particular, the high-K concentration causes depolarisation of vascular smooth muscle and epithelial cell membrane, and can lead to blood vessel constriction and rapid cellular swelling. In the first part of the present study, our results demonstrate that K-UW, but not Na-UW, induced a reproducible increase in vascular resistances of the IPK. To our knowledge these results are the first to clearly illustrate a vaseactive effect of high-potassium cold-storage solutions. In the second part of this study, we tried to inhibit vasoconstriction by adding nifedipine to the cold-storage solutions. The addition of nifedipine to K-UW produced a significant reduction of the peak perfusion pressure, while Na-UW infusion was not influenced by this CEB. In the third part of the study, we evaluated the functional recovery of kidneys after 24-hour cold storage in K-UW and Na-UW with or without nifedipine. In control IPK (without cold storage), infusion of nifedipine did not modify hemodynamics or tubular function. Under conditions of in vitro perfusion with perfusion pressure of 80 to 100 mm Hg, the IPK appears to possess little intrinsic vascular resistance, and in the absence of exogenous vasocontrictors, CEB exert no effect on renal perfusate flow or glomerular filtration rate in this model. 7 As we and others have demonstrated previously, cold storage of kidneys for 24 hours results in a considerable alteration in GFR and tubular function as assessed by FRNa
CALCIUM ANTAGONISTS
and ARNa in the IPK, no matter the composition of the cold-storage solution is,3'7'8 and we confirmed that Na-UW appears slightly more effective than the original highpotassium U W solution. These results have been recently validated in the isogenic graft model in the rat. 9 Other authors reported similar results by using other experimental animals or other low K-high Na solutions. 1'2 Finally, we added nifedipine to K-UW and Na-UW during flush and storage for 24 hours, and then to the normothermic reperfusate of the kidney. Nifedipine did not induce any significant change in GFR, or in tubular function or hemodynamics in IPK cold stored in K-UW. In contrast, the addition of nifedipine to Na-UW improved both G F R and tubular function of the cold-stored kidney, while Na-UW without nifedipine ameliorated only tubular function. CONCLUSION
Nifedipine may be of potential effect in preventing or attenuating ischemic injury by a mechanism that does not involve its vasodilatory properties. This drug may interfere with the hypoxic-induced disturbance in cellular calcium
2441
metabolism or act by other mechanisms such as inhibition of ion-channel activities or prevention of cell swelling. Further studies are needed to clearly evaluate the potential clinical role of CEB in the prevention of ischemic acute renal failure posttransplantation.
REFERENCES
1. Moen J, Claesson K, Pienaar H, et al: Transplantation 47:940, 1989 2. Bizugas M, Jablonski P, Thomas AC, et al: Transplantation 49:872, 1990 3. Ramella S, Hadj-Aissa A, Barbieux A, et al: Nephrol Dial Transplant 75:842, 1995 4. Dawidson I, Rooth P, Lu C, et al: J Am Soc Nephrol 2:983, 1991 5. Palmer B, Dawidson I, Sagalowsky, et al: Transplantation 52:640, 1991 6. Bonventre J: Kidney Int 43:1160, 1993 7. Loutzenhiser R, Horton C, Epstein M: Nephron 39:382, 1985 8. Marshall V, Ross B, Bishop M, et al: Transplantation 26:315, 1978 9. Feitosa Tajra LC, Ramella-Virieux S, Benabdennebi H, et al: Transplant Proc 28:2905, 1996
Calcium Antagonists Improve Kidney Function in the Rat After Cold Storage in High-Na UW but Not in High-K UW Solution A. Hadj-ATssa, S.C. Ramella-Virieux, J.P. Steghens, A. Barbieux, and N. Pozet X T R A C E L L U L A R types (high-Na) of cold-storage solution (CSS) have been shown to be more effective in preserving kidneys than intracellular CSS (high-K). 1"2We have previously demonstrated with the isolated perfused rat kidney model (IPK) that a high-Na version of University of Wisconsin (UW) Belzer's CSS (Na-UW) seemed to be less injurious to recovery function than the original high-K UW Belzer's solution (K-UW). 3 On the other hand, a number of clinical investigations have suggested that calcium entry blockers (CEB) may improve graft function when administered after and/or prior to transpiantation. 4"5 The ischemia reperfusion syndrome involves, in part, an alteration in intracellular calcium metabolism that induces an increase in renal vascular resistances (RVR) and other cellular dysfunction, and high-K CSS per se are vasoconstrictive.6 Since maneuvers that would prevent vasoconstriction may favour the intraorgan diffusion of the cold-storage solution and ameliorate preservation, we evaluated with the IPK model, the actual benefit of the vasodilator nifedipine on kidneys preserved in K-UW and Na-UW.
E
MATERIALS AND METHODS The IPK Technique
Sprague-Dawley rats (weighing 200 to 250 g) were used. Unless otherwise indicated, the technique for IPK has been previously described in detail.3 Cold-Storage Solutions
Two CSS, derived from UW Belzer's solution and prepared in the pharmaceutical department,3 were compared: (1) K-UW solution was identical to the original Belzer's high-potassium UW solution (125 mmol/L potassium and 30 mmol/L sodium); and (2) Na-UW solution contained the same agents as the original UW solution but with high-sodium and low-potassium concentrations (125 mmol/L sodium and 30 mmol/L potassium). EXPERIMENTAL PROTOCOLS AND RESULTS
The IPK was used, first as a vascular bed to test the effects of CSS on renal vascular resistances and the influence of nifedipine. Second, the recovery function of the IPK was assessed by glomerular filtration rate (GFR, inulin clearance) and tubular Na reabsorption, after 24-hour preservation in K-UW and Na-UW, with or without nifedipine. Results were compared with a control group in which renal function was measured without prior cold storage. © 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Transplantation Proceedings, 29, 2439-2441 (1997)
Effects of Cold-Storage Solutions on Renal Vascular Resistances of the IPK
The kidneys were placed in the isolated perfusion circuit and perfused with Krebs-Henseleit solution without recirculation of the effluent, at a rate of 10 mL/min until stabilization of renal perfusion pressure. Therefore, the CSS was infused through the main perfusate line during 1 minute. When the CSS infusion was started, the perfusion of Krebs-Henseleit was stopped so that the kidney was constantly perfused at a rate of 10 mL/min. As a result, recorded changes in perfusion pressure are actually in relation to changes in RVR and not in the rate of perfusion. For each cold-storage solution, four rats were used. The infusion of K-UW induced significant increases in perfusion pressure of 90 _+ 20 mm Hg. Conversely, an identical infusion rate of Na-UW did not induce any significant change in perfusion pressure (+9.5 _+ 0.6 mm Hg). Effects of Nifedipine on the Changes in RVR Induced by the CSS
After cannulation of the kidney and its perfusion with Krebs-Henseleit solution as described above, the CSS was infused during 1 minute every 5 minutes for 10 times, at a rate of 10 mL/min. When the CSS was started, the KrebsHenseleit perfusion was stopped. After the CSS had been infused for three consecutive times, to confirm the reproducibility of the induced changes in perfusion pressure, an infusion of nifedipine (10 6 mol/L in NaCI 0.9%) was added during 10 minutes and then withdrawn. For each CSS, four rats were used. Before adding nifedipine, the infusion of K-UW for three consecutive times induced a reproducible increase in perFrom the Department of Renal Functional Exploration (A.H.-A., S.G.R.-V., N.P.), Laboratory of Biochemistry C (J.P.S.), and Pharmaceutical Department (A.B.), Edouard Herriot Hospital and Claude Bernard University, Lyon, France. Supported by the Conseil General du Rh6ne and by grants from the Hospices Civils de Lyon and the Fondation de rAvenir, France. Address reprint requests to Dr A. Hadj-Aissa, Service d'Exploration Fonctionnelle Renale, H6pital Edouard Herriot, Pavilion P, 69437 Lyon Cedex 03, France. 0041-1345/97/$17.00 PII S0041-1345(97)00441-7 2439
2440
HADJ-AiSSA, RAMELLA-VIRIEUX, STEGHENS ET A L
Table 1. Glomemlar Filtration Rate (GFR), Fractional Na Reabsorption (FR.a), Absolute Na Reabsorption (AR..), Perfusion Pressure (PP), and Renal Vascular Resistances (RVR) in Control IPK and in Kidneys After 24 Hours of Cold Storage in K-UW and Na-UW, With (+) or Without (-) addition of Nifedipine Control Nifedipine (-) GFR (/zL/rnin/g) FRN, (%) ARNa (/zmol/min/g) PP (mm Hg) RVR (mm Hg/mL/min/g)
441 91.4 55.48 99.7 5.3
-- 54 -+ 1.7 _+ 6.14 -+ 0.8 -+ 0.7
K-UW Nifedipine (+)
305 86.9 37.90 98.8 5.1
-+ 51 _+ 3.2 _+ 7.34 -+ 1.8 _+ 0.6
Na-UW
Nifedipine (-)
Nifedipine (+)
49 26.7 2.26 97.4 5.3
41 34.4 2.32 98.0 5.8
+ t1" _+ 1.9" _+ 0.69* _+ 0.35 -+ 0.5
_+ 13" _+ 7.4* _+ 1.19" _+ 2.4 _+ 0.6
Nifedipine (-) 52 58.2 4.61 99.7 5.8
_+ 10" _+ 7.4 *t _ 0.97 *t _+ 1.4 _+ 0.5
Nifedipine (+) 129 74.2 13.04 99.2 4.9
+ 21 *it _+ 4.9 *t _+ 2.15 *tt _+ 0.7 _+ 0.4
*P < .05 vs control. tp < .05 vs K-UW. tP < .05 vs nifedipine (-).
fusion pressure of 54 ± 13 mm Hg above base-line. In similar conditions, the infusion of Na-UW induced a slight, not significant, decrease in perfusion pressure of 8 - 3 mm Hg below base-line. The addition of nifedipine to K-UW produced a significant reduction of the peak perfusion pressure to only 17 -+ 6 mm Hg, whereas Na-UW infusion was not influenced by nifedipine ( - 3 -+ 1 mm Hg).
stored with K-UW. In contrast, compared to Na-UW N - , the addition of nifedipine to kidneys stored in Na-UW induced a significant increase in GFR and ARNa. FRNa increased, but not significantly. Table 1 shows that renal function of Na-UW-nifedipine became significantly higher than that of K-UW N+. There was no difference in PP or RVR between K-UW, Na-UW, and control.
Influence o f Nifedipine on Renal F u n c t i o n o f t h e IPK (Table 1)
DISCUSSION
Without Nifedipine (N-). In the control group N - (n = 6), the kidneys were perfused normothermically with the Krebs-Henseleit-albumine solution immediately after they were harvested. Results are shown in Table 1. In the cold-stored group N - , kidneys were catheterized and isolated without interruption of blood flow and flushed with the cold-storage solution cooled at +4°C for 10 minutes at 3 mL/min. The kidneys were then placed with their catheters in a small container with 30 mL of the cold-storage solution and stored at 0-4°C for 24 hours. Afterward, the organs were reperfused on the isolated perfusion circuit with perfusion medium at 37°C to measure renal function and hemodynamics. In these experiments 14 rats were used, 7 for each cold-storage solution. After 24 hours of cold storage in each of the two solutions, GFR was dramatically decreased by 90% compared to control. Na reabsorption was also considerably altered by cold storage. However, both fractional Na reabsorption (FRNa) and absolute Na reabsorption (ARNa) of kidneys preserved in Na-UW were significantly higher than in K-UW. There was no difference in PP or RVR between cold-stored kidneys and control. With Nifedipine (N+). The same experiments were performed as indicated above, but nifedipine 10 - 6 mol/L was added to the cold-storage solution and to the KrebsHenseleit perfusion medium. In these last experiments, 18 rats were used: six controls and six for each cold-storage solution. In the control group N+, the addition of nifedipine did not cause any significant alteration in the functional parameters (Table 1). In the cold-stored group N+, nifedipine did not significantly influence renal function of kidneys flushed and
Cold-storage solutions commonly used for preservation of organs before transplantation such as Euro-Collins (EC) and Belzer's UW solutions have a high potassium concentration. These solutions are supposed to limit potassium leakage from stored cells and thereby maintain a more normal intracellular milieu. However, high potassium-containing solutions have several disadvantages. In particular, the high-K concentration causes depolarisation of vascular smooth muscle and epithelial cell membrane, and can lead to blood vessel constriction and rapid cellular swelling. In the first part of the present study, our results demonstrate that K-UW, but not Na-UW, induced a reproducible increase in vascular resistances of the IPK. To our knowledge these results are the first to clearly illustrate a vaseactive effect of high-potassium cold-storage solutions. In the second part of this study, we tried to inhibit vasoconstriction by adding nifedipine to the cold-storage solutions. The addition of nifedipine to K-UW produced a significant reduction of the peak perfusion pressure, while Na-UW infusion was not influenced by this CEB. In the third part of the study, we evaluated the functional recovery of kidneys after 24-hour cold storage in K-UW and Na-UW with or without nifedipine. In control IPK (without cold storage), infusion of nifedipine did not modify hemodynamics or tubular function. Under conditions of in vitro perfusion with perfusion pressure of 80 to 100 mm Hg, the IPK appears to possess little intrinsic vascular resistance, and in the absence of exogenous vasocontrictors, CEB exert no effect on renal perfusate flow or glomerular filtration rate in this model. 7 As we and others have demonstrated previously, cold storage of kidneys for 24 hours results in a considerable alteration in GFR and tubular function as assessed by FRNa
CALCIUM ANTAGONISTS
and ARNa in the IPK, no matter the composition of the cold-storage solution is,3'7'8 and we confirmed that Na-UW appears slightly more effective than the original highpotassium U W solution. These results have been recently validated in the isogenic graft model in the rat. 9 Other authors reported similar results by using other experimental animals or other low K-high Na solutions. 1'2 Finally, we added nifedipine to K-UW and Na-UW during flush and storage for 24 hours, and then to the normothermic reperfusate of the kidney. Nifedipine did not induce any significant change in GFR, or in tubular function or hemodynamics in IPK cold stored in K-UW. In contrast, the addition of nifedipine to Na-UW improved both G F R and tubular function of the cold-stored kidney, while Na-UW without nifedipine ameliorated only tubular function. CONCLUSION
Nifedipine may be of potential effect in preventing or attenuating ischemic injury by a mechanism that does not involve its vasodilatory properties. This drug may interfere with the hypoxic-induced disturbance in cellular calcium
2441
metabolism or act by other mechanisms such as inhibition of ion-channel activities or prevention of cell swelling. Further studies are needed to clearly evaluate the potential clinical role of CEB in the prevention of ischemic acute renal failure posttransplantation.
REFERENCES
1. Moen J, Claesson K, Pienaar H, et al: Transplantation 47:940, 1989 2. Bizugas M, Jablonski P, Thomas AC, et al: Transplantation 49:872, 1990 3. Ramella S, Hadj-Aissa A, Barbieux A, et al: Nephrol Dial Transplant 75:842, 1995 4. Dawidson I, Rooth P, Lu C, et al: J Am Soc Nephrol 2:983, 1991 5. Palmer B, Dawidson I, Sagalowsky, et al: Transplantation 52:640, 1991 6. Bonventre J: Kidney Int 43:1160, 1993 7. Loutzenhiser R, Horton C, Epstein M: Nephron 39:382, 1985 8. Marshall V, Ross B, Bishop M, et al: Transplantation 26:315, 1978 9. Feitosa Tajra LC, Ramella-Virieux S, Benabdennebi H, et al: Transplant Proc 28:2905, 1996