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Amelioration of renal ischemic injury by phosphocreatine
JOURNAL
OF SURGICAL
RESEARCH
Amelioration
51,2’71-274
(1991)
of Renal lschemic
Injury by Phosphocreatine’
RICARDORODRIGUEZ, M.D., MICHAEL~TEPKE, M.D., STACIEMAITZ, B.S., CHARLES B. CUONO, M.D., PH.D., AND BAUER E. SUMPIO, M.D., PH.D.’ Department
of Surgery,
Yale University Submitted
School of Medicine,
for publication
November
06510~8062
20, 1990
at counteracting the effects of oxygen free radicals [2]. This strategy entails either the prevention of free radical formation or the removal of these injurious agents by the use of free radical scavengers. A third line of investigation has been directed at maintaining or restoring phosphometabolite balance within the cell [3,4]. Several investigators have shown a clear correlation between ATP levels or total adenine nucleotide content in predicting organ survival [ 51. We hypothesize that maintenance or early restoration of phosphometabolite balance within the cells of the renal parenchyma may be an important step in preventing ischemic injury. This hypothesis was based on previous work with skin flaps. Using 31P and ‘H NMR Cuono et al. established the pivotal role of phosphocreatine in sustaining adenine nucleotide levels in skin and demonstrated that depletion of phosphocreatine correlates with flap necrosis [6]. They have also previously reported that parenteral administration of phosphocreatine following elevation of experimental skin flaps significantly enhances skin flap survival [7]. The aim of this present study was to determine the efficacy of phosphocreatine-containing perfusates in preservation of renal function in an established experimental model of kidney ischemia [ 41.
Phosphocreatine (PCr) is a critical intracellular energy reservoir used in the regeneration of ATP. The aim of this study was to determine the efficacy of exogenously added PCr on preservation of renal function in an in vitro model. The renal artery and ureter of a rat were cannulated and the kidney was subjected to 45 min of normothermic in vivo ischemia. The kidneys were then perfused ex vivo with either a Krebs-bicarbonate solution (Krebs) or a Krebs solution containing 3 n&f PCr or an osmotically balanced solution containing 3 mM PCr. Our results indicate that the perfusion of kidneys subjected to 45 min of warm ischemia with solutions containing PCr resulted in significant improvements in GFR, RPF, and V, FRN, and FRazo compared to KREBS alone. This suggests that the important factor in preservation of kidney function after an initial ischemic insult may be the addition of PCr o issl Academic rather that the electrolyte solution used. Press,
New Haven, Connecticut
Inc.
Ischemia-induced organ injury is usually exacerbated with the reestablishment of blood flow. This “reperfusion injury” consists of a constellation of biochemical events resulting in cell edema and cell membrane disruption. Attempts to mitigate this injury have been centered around three areas of investigation. The first area of investigation is prevention of intracellular swelling and acidosis by establishing the optimal composition of solutions used to perfuse the ischemic organ. Attempts at mimicking the “intracellular milieu” or improving the buffering capacity of the electrolyte solutions have instigated considerable debate as to the relative merits of these manuevers. Jamieson and colleagues have demonstrated that reperfusion with a solution containing impermeant anions (raffinose and lactobionate) was successful in preserving livers ex vivo for extended periods in hypothermic conditions [l]. Another approach at reducing reperfusion injury has been directed
MATERIALS
AND METHODS
Isolated Perfused Rat Kidney Preparation Male Holtzman rats 250-300 g were used as kidney donors for the isolated perfused right kidney preparation. Rats were fasted overnight but allowed water ad lib. They were anesthetized with pentobarbital sodium (50 mg/kg ip) before experimentation. Kidneys were isolated as described in detail previously [4]. The right ureter was cannulated with PE 10 tubing and after systemic heparinization (500 units/kg iv) an 18-gauge needle was introduced into the right renal artery and flow immediately initiated from a gravity reservoir to maintain perfusion of the kidney while it was dissected free of its connective tissue attachments as well as to flush the kidney of blood. The kidney was then perfused in an oxygenated closed circuit apparatus at 37°C with a nonpulsatile pump, with the net mean renal arterial pres-
1 Presented at the Annual Meeting of the Association for Academic Surgery, Houston, TX, November 14-17,199O. ‘To whom correspondence and reprint requests should be addressed at Department of Surgery, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, Connecticut 06510-8062. 271
All
Copyright 0 1991 rights of reproduction
0022.4804/91$1.50 by Academic Press, Inc. in any form reserved.
272
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
51, NO. 4, OCTOBER
1991
Statistics
ml/min 160
CONTROL KREBS PCr-KREBS PCr+R-SOL
1
GFR (1043
u
RPF
FIG. 1. Effects of reperfusion of kidneys after 45 min of normothermic ischemia. Kidneys were subjected to 45 min of warm ischemia, then reperfused with solutions of Krebs, phosphocreatine added to Krebs (PCr-Krebs), and phosphocreatine added to a modified “UW” solution (PCr + R-SOL) (see Methods for details). Glomerular filtration rate (GFR), urine flow (II), and renal perfusate flow (RPF) were measured and the mean f SE (n = 6 kidneys) was determined and compared to values obtained from nonischemic kidneys perfused with Krebs (control); *P < 0.05 compared to control, +P < 0.05 compared to Krebs.
sure continually adjusted to 100 mm Hg (control, nonischemic kidneys, n = 6). In other experiments kidneys were then subjected to 45 min of warm ischemia with the abdomen closed and no flow through the cannula (normothermic ischemic kidneys). Following this period of ischemia the kidney was removed with its vascular pedicle stump and ureter intact and perfused as described above. Experimental Protocol Kidneys subjected to 45 min of normothermic ischemia were perfused ex vivo for 120 min with one of three solutions. The first (Krebs, n = 6) was a Krebs-bicarbonate solution containing 7.5 g/dl of bovine serum albumin, 1 mg/ml glucose, 0.5 mg/ml creatinine, a mixture of amino acids, and trace amounts of [3H]inulin (glomerular marker). In the second solution (PCr + Krebs, n = 6), 3 mM phosphocreatine was added to the Krebs bicarbonate solution. The third solution was a modification of the “UW” solutions (PCr + R-SOL, n = 6) and consisted of 100 mM K+ lactobionate, 2.5 mM NaKH,PO,, 30 n&f raffinose, 5 mA4 MgSO,, 3 mM glutathione, 3 mM phosphocreatine, and trace amounts of [3H]inulin (osmolarity, 300-310 mOsm/liter). After an initial 10 min equilibration period, urine and perfusate samples were collected at 10 min clearance intervals. Perfusate flow was recorded from a Brooks flow meter in series with the arterial cannula. Urine volumes (V) were determined gravimetrically by collecting the urine in preweighed vials. Aliquots of perfusate (P) and urine (U) were placed in scintillation fluid (Aquasol) and analyzed for 3H radioactivity. Sodium (Na+) was determined by flame spectrophotometry (Turner Model 510). Glomerular filtration (GFR, ml/min) for each timed interval was calculated from the clearance of [3H]inulin ([U/P]in X V). The fractional reabsorption of water was calculated using inulin as a marker (l-[P/ U-L,, UJin). The fractional reabsorption of Na(FR,,) was calculated using the formula (l-[U/PJNa/[U/P] in).
Data are expressed as means f SE. Paired and unimpaired Student t test was utilized wherever appropriate. P < 0.05 was considered significant. RESULTS The results seen in Figs. 1 and 2 are given for nonischemic control kidneys (CONTROL) and ischemic kidneys perfused with either Krebs, PCr Krebs, or PCr + R- Sol. As shown in Fig. 1, kidneys subjected to 45 min of normothermic ischemia and subsequently perfused with Krebs solution were oliguric and had very low initial GRF with poor recovery. Kidneys treated with both Krebs + PCr solution and PCr + R-Sol had higher initial GFR values. The improvement in GFR values was sustained, reaching up to 80% of control values. Ischemic kidneys reperfused with phosphocreatine solutions showed high flow rates which where significantly improved over control values. These high flow rates were sustained through of 120 min of perfusion. In addition, urine flow rates were significantly enhanced with both phosphocreatine-containing solutions. The PCr + R-Sol perfused kidneys exhibited the highest urine flow rate, attaining values of up to 3 times control. The fractional reabsorption of sodium in the two PCr containing solutions were significantly improved over Krebs solution alone and closely approximated control values (see Fig. 2). In contrast fractional reabsorption of water differed in the PCr-KREBS solution compared with the PCr + R-Sol. The PCr + Krebs solution had slightly higher values for fractional reabsorption of water. DISCUSSION In general, experimental approaches to amelioration of renal ischemic injury have focused on the prevention or attenuation of intracellular swelling, prevention of
CONTROL KREBS PCr-KREBS PCr+R-SOL
FRN,
FR yo
FIG. 2. Effects of reperfusion of kidneys after 45 min of normothermic ischemia. Kidneys were subjected to 45 min of warm ischemia, then reperfused with solutions of Krebs, phosphocreatine added to Krebs (PCr-Krebs), and phosphocreatine added to a modified “WV” solution (PCr + R-sol) (see Methods for details). Fractional excretion of sodium (FR,,) and fractional excretion of water (FRH,O) were measured and the mean -C SE (n = 6 kidneys) was determined and compared to values obtained from nonischemic kidneys perfused with Krebs (control); +P c 0.05 compared to Krebs.
RODRIGUEZ
ET AL.: PHOSPHOCREATINE
IN RENAL
273
ISCHEMIA
Ca++ XANTHINE DEHYROGENASE (Ca++ PROTEASE)
k INOSINE
AMP HYPOXANTHINE
P PCr Cr
A N I E
REPERFUSION
i)
FIG. 3. As ischemic time increases ATP is progressively phosphorylated to AMP; once AMP is dephosphorylated to adenosine, adenosine leaks out of the cell. Adenosine is then metabolized to inosine and hypoxanthine, which in the presence of zanthine oxidase will react with reoerfused oxygen _- to create oxygen free radicals. In addition, the cell membrane becomes incompetent due to electrolyte pump failure permitting the influence of both sodium and calcium ions.
injury mediated through oxygen free radicals and removal (scavenging) of such agents, and the use of exogenous substrates to maintain the level of high energy phosphometabolites and total adenine nucleotide content in the cell. These three approaches address a continuum of changes shown diagramatically in Fig. 3. The increased tendency for cell swelling after reperfusion constitutes the basis for the use of perfusates during ischemia which mimic the “intracellular milieu.” One of the most efficacious has been the “UW solution.” This is a slightly hypoosmolar (300 to 310 mOsm) electrolyte solution containing raffinose (30 m&f) and lactobionate (100 mM). Raffinose is an impermeable trisaccharide and lactobionate is an impermeable disaccharide anion. Both raffinose and lactobionate have been proven to be much more effective impermeants than other substrates such as sodium citrate. This solution, while beneficial in organ preservation, does not address the issue of energy depletion (ATP dephosphorylation) or the leakage of adenosine from the cytosol. One approach has been to give high concentrations of PO, to inhibit adenosine deaminase, as well as providing high levels of adenosine in the perfusate to facilitate ATP synthesis [8]. The main problem with these agents is that they are rapidly degraded by cell membrane enzymes. Therefore, increasing the concentration of adenosine extracellularly may not result in osmotic equilibration across the cell membrane. Rather, there will be increased concentration of inosine and hypoxantine, its metabolic products. The mechanisms of “free radical” damage created by the increasing concentrations of inosine and hypoxantine are well described. Needless to say there have been many attempts at ameliorating renal ischemic injury by use of free oxygen radicals scavengers. These studies, however, have met with mixed results.
Phosphocreatine is the major high energy phosphometabolite of a number of organs including the brain, skeletal muscle, heart, and skin [9]. Its main theoretic advantages are its ability to penetrate the cell membrane and the potential for the high energy phosphate group to be translocated by cell membrane kinases, normally found in mitrochondria [lo]. Thus providing exogenous phosphocreatine to ischemic tissues may provide a readily available source of high energy phosphate to be used in the resynesthesia of ATP. Since ATP is not freely interchangeable but is compartmentalized to different parts of the cell as membrane-bound ATP-kinase complexes, phosphocreatine functions as the “energy currency” [ 111 of the cell delivering needed high energy phosphates to different membrane-bound ATP kinase complexes. Furthermore, the creatine base serves to buffer the production of hydrogen ions that occurs during ischemia. In the cytoplasm, the ATP pool is preserved at the expense of phosphocreatine. The maintenance of a pool of phosphorylated adenine nucleotides will effectively prevent leakage of adenosine. This will decrease the pool of reagents necessary for free oxygen radical production (inosine and hypoxanthine). Although the efficacy of phosphocreatine added to solutions perfusing skin flap [7] or heart [12] models have been reported, the role of phosphocreatine in the kidney has not been well delineated. Previous 31PNMR spectroscopy studies have detected low levels of phosphocreatine in the kidney [13]. In addition, whole renal tissue levels of creatine kinases have been reported as low, composed exclusively of the “brain” isozyme but not the “muscle type” [lo]. These observations no doubt are responsible for the relative paucity of studies addressing the role of phosphocreatine in recovery of postischemic renal injury.
274
JOURNAL
OF SURGICAL
RESEARCH:
More recently, enzymatic assays have confirmed the presence of significant levels of phosphocreatine in the kidneys [14]. Further, this phosphocreatine pool responds predictably to ischemic insult. Phosphocreatine concentration in the kidney appears to vary, ranging from about 2 mmole/kg (dry weight) in the proximal straight tubule, thick ascending Loop of Henle, and papilla to about 10 mmole/kg in the distal convoluted tubule. The level of phosphocreatine in the distal convoluted tubule is comparable to that in brain. In comparison, ATP levels in proximal tubule segments are about 10 mmole/kg (dry weight) and 16 to 17 mmole/kg (dry weight) in the distal tubule segments. Renal creatine kinase activity varies regionally loo-fold. Measured levels were highest in the distal convoluted tubule and lowest in the proximal convoluted tubule. In the thick ascending Loop of Henle as well as in the distal convoluted tubule, phosphocreatine is thought to be consumed at the expense of ATP 1141. If indeed, phosphocreatine does play a role in energy metabolism in the kidney, exogenously administered phosphocreatine after a period of ischemia should help in the functional recovery of the kidney. In this study, we compared a solution which closely approximates the “UW” solution in that it contains the impermeant anions lactobionate and raffinose, the free radical scavenger, glutathione, and the energy substrate, phosphocreatine. This solution was compared both to a standard electrolyte solution (Krebs) as well as Krebs containing phosphocreatine. If prevention of intracellular swelling is the main determinant in ameliorating renal ischemic injury, one would expect to observe a significant difference between ischemic kidneys reperfused with the PCr + R-Sol solution and those reperfused with PCr + Krebs solution. No such differences were observed. Previous studies utilizing ATP/magnesium chloride in the reperfusion of ischemic kidneys demonstrated a beneficial effect on renal function but with progressive diuresis [4]. This was also observed in our PCr-treated ischemic kidneys. The increase in urine flow rate was more marked in the PCr + R solution and was associated with a slightly decreased (but not statistically significant) fractional reabsorption of water. The increase in urine flow rate and the decreased fractional reabsorption of water in the PCr + R solution may be due to the presence of the impermeant anions raffinose and lactobionate which when present in the tubular urine may diminish the passive reabsorption of water. While this effect might be beneficial in cold storage preservation of kidneys, in this study it did not result in any significant difference in amelioration of renal ischemic injury as compared to the Krebs + PCr solution. The fractional resorption of sodium, however, was improved for both the PCr + Krebs solution and the PCr + R solutions. This is an energy-requiring step and suggests that intracellular energy stores were replenished.
VOL.
51, NO. 4, OCTOBER
1991
The findings of this study establish that phosphocreatine has beneficial effects on the recovery of renal function following ischemia. The mechanism of this effect appears to be independent of the electrolyte composition of the solution. Use of an electrolyte solution which was designed to diminish intracellular swelling did not add any substantial benefit to that obtained by phosphocreatine in combination with a standard electrolyte solution. It suggests that phosphocreatine ameliorates renal ischemic injury by repletion of phosphometabolite balance and may prevent adenosine leakage which leads to “free radical” generation. ACKNOWLEDGMENT This work was supported in part by an OHSE Grant from the Department of Surgery, Yale University School of Medicine.
REFERENCES 1.
2. 3.
4.
5.
6.
7.
a.
9. 10. 11.
12.
13.
14.
Jamieson, N. V., Lindell, S., Sundberg, R., Southard, J. H., and Belzer, F. D. An analysis of the components in U.W. solution using the isolated perfused rabbit. Liver Transplun. 46: 512, 1988. McCord, J. M. Free Radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science 186: 5291974. Collins, G. M., Taft, P., Green, R. D., Ruprecht, R., and Halasz, V. A. Adenine nucleotide levels in preserved and ischemically injured canine kidneys. World J. Surg. 1: 237,1977. Sumpio, B. E., Chaudry, I. H., Clemens, M. G., and Baue, A. E. Accelerated functional recovery of isolated rat kidney with ATPMgCl, after warm ischemia. Am. J. Physiol247 (Regulatory Intergrature Comp. Physiol): R:1047-R1053, 1984. Nichida, T., Koseki, M., Kanuike, W., Nakahara, M., Nakao, K., Kawashima, Y., Hashimoto, T., and Tagawa, K. Levels of purine compounds in a perfusate as a biochemical marker of ischemic injury of cold preserved liver. Tansplantation 44(l): 16,1987. Cuono, C. B., Armitage, I. A., Marquefand, B. S., and Chapo, G. L. Nuclear Magnetic Resonance Spectroscopy of Skin: Predictive correlates for clinical application. Plast. Reconstr. Surg. 81(l): 1, 1988. Cuono, C. B., Cartes, A., Marguetand, R., Weisberg, V., and Armitage, I. M. Skin flap survival via postoperative phosphocreatine replacement. Surgery 102(2): 263, 1988. Belzer, F. O., Sollinger, H. W., Glass, N. R., Miller, D. T., Hoffman, R. M., and Southard, J. H. Beneficial effects of adenosine and phosphate in preservation. Transplantation 36(6): 633, 1983. Bessman, S. P., and Carpenter, C. L. The creatine phosphate energy shuttle. Ann. Rev. Biochem. 64: 831,1985. Davison, D. M., and Fine, I. H. Creatine kinase in human tissues. Arch. Neural. 16: 175, 1967. Saks, V. A., Llpina, N. V., Sharov, V., Smirnov, V. I., Chazon, E., and Grosse, R. Role of creatine phosplokinase in cellular function and metabolism. Can. J. Physiol. Pharmacol. 56: 691, 1978. Robinson, L. A., Braimbridge, M. V., and Hearse, D. J. Creatine phosphate: An additive myocardial protective and antiarrythnic agent in cardioplegia. J. Thorac. Cardiuvasc. Surg. 87: 190,1984. Sehr, P. A., Radda, G. K., Bore, P. J., and Sells, R. A., et al. A model kidney transplant studied by phosphorous nuclear resonance. Biochem. Biophys. Res. Commun. 77: 195, 1984. Bastine, J., Cambon, N., Thompson, M., Lowry, 0. H., and Burch, H. B. Change in energy reserves in different segments of the nephron during brief ischemia. Kidney Int. 31: 1239,1987.
OF SURGICAL
RESEARCH
Amelioration
51,2’71-274
(1991)
of Renal lschemic
Injury by Phosphocreatine’
RICARDORODRIGUEZ, M.D., MICHAEL~TEPKE, M.D., STACIEMAITZ, B.S., CHARLES B. CUONO, M.D., PH.D., AND BAUER E. SUMPIO, M.D., PH.D.’ Department
of Surgery,
Yale University Submitted
School of Medicine,
for publication
November
06510~8062
20, 1990
at counteracting the effects of oxygen free radicals [2]. This strategy entails either the prevention of free radical formation or the removal of these injurious agents by the use of free radical scavengers. A third line of investigation has been directed at maintaining or restoring phosphometabolite balance within the cell [3,4]. Several investigators have shown a clear correlation between ATP levels or total adenine nucleotide content in predicting organ survival [ 51. We hypothesize that maintenance or early restoration of phosphometabolite balance within the cells of the renal parenchyma may be an important step in preventing ischemic injury. This hypothesis was based on previous work with skin flaps. Using 31P and ‘H NMR Cuono et al. established the pivotal role of phosphocreatine in sustaining adenine nucleotide levels in skin and demonstrated that depletion of phosphocreatine correlates with flap necrosis [6]. They have also previously reported that parenteral administration of phosphocreatine following elevation of experimental skin flaps significantly enhances skin flap survival [7]. The aim of this present study was to determine the efficacy of phosphocreatine-containing perfusates in preservation of renal function in an established experimental model of kidney ischemia [ 41.
Phosphocreatine (PCr) is a critical intracellular energy reservoir used in the regeneration of ATP. The aim of this study was to determine the efficacy of exogenously added PCr on preservation of renal function in an in vitro model. The renal artery and ureter of a rat were cannulated and the kidney was subjected to 45 min of normothermic in vivo ischemia. The kidneys were then perfused ex vivo with either a Krebs-bicarbonate solution (Krebs) or a Krebs solution containing 3 n&f PCr or an osmotically balanced solution containing 3 mM PCr. Our results indicate that the perfusion of kidneys subjected to 45 min of warm ischemia with solutions containing PCr resulted in significant improvements in GFR, RPF, and V, FRN, and FRazo compared to KREBS alone. This suggests that the important factor in preservation of kidney function after an initial ischemic insult may be the addition of PCr o issl Academic rather that the electrolyte solution used. Press,
New Haven, Connecticut
Inc.
Ischemia-induced organ injury is usually exacerbated with the reestablishment of blood flow. This “reperfusion injury” consists of a constellation of biochemical events resulting in cell edema and cell membrane disruption. Attempts to mitigate this injury have been centered around three areas of investigation. The first area of investigation is prevention of intracellular swelling and acidosis by establishing the optimal composition of solutions used to perfuse the ischemic organ. Attempts at mimicking the “intracellular milieu” or improving the buffering capacity of the electrolyte solutions have instigated considerable debate as to the relative merits of these manuevers. Jamieson and colleagues have demonstrated that reperfusion with a solution containing impermeant anions (raffinose and lactobionate) was successful in preserving livers ex vivo for extended periods in hypothermic conditions [l]. Another approach at reducing reperfusion injury has been directed
MATERIALS
AND METHODS
Isolated Perfused Rat Kidney Preparation Male Holtzman rats 250-300 g were used as kidney donors for the isolated perfused right kidney preparation. Rats were fasted overnight but allowed water ad lib. They were anesthetized with pentobarbital sodium (50 mg/kg ip) before experimentation. Kidneys were isolated as described in detail previously [4]. The right ureter was cannulated with PE 10 tubing and after systemic heparinization (500 units/kg iv) an 18-gauge needle was introduced into the right renal artery and flow immediately initiated from a gravity reservoir to maintain perfusion of the kidney while it was dissected free of its connective tissue attachments as well as to flush the kidney of blood. The kidney was then perfused in an oxygenated closed circuit apparatus at 37°C with a nonpulsatile pump, with the net mean renal arterial pres-
1 Presented at the Annual Meeting of the Association for Academic Surgery, Houston, TX, November 14-17,199O. ‘To whom correspondence and reprint requests should be addressed at Department of Surgery, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, Connecticut 06510-8062. 271
All
Copyright 0 1991 rights of reproduction
0022.4804/91$1.50 by Academic Press, Inc. in any form reserved.
272
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
51, NO. 4, OCTOBER
1991
Statistics
ml/min 160
CONTROL KREBS PCr-KREBS PCr+R-SOL
1
GFR (1043
u
RPF
FIG. 1. Effects of reperfusion of kidneys after 45 min of normothermic ischemia. Kidneys were subjected to 45 min of warm ischemia, then reperfused with solutions of Krebs, phosphocreatine added to Krebs (PCr-Krebs), and phosphocreatine added to a modified “UW” solution (PCr + R-SOL) (see Methods for details). Glomerular filtration rate (GFR), urine flow (II), and renal perfusate flow (RPF) were measured and the mean f SE (n = 6 kidneys) was determined and compared to values obtained from nonischemic kidneys perfused with Krebs (control); *P < 0.05 compared to control, +P < 0.05 compared to Krebs.
sure continually adjusted to 100 mm Hg (control, nonischemic kidneys, n = 6). In other experiments kidneys were then subjected to 45 min of warm ischemia with the abdomen closed and no flow through the cannula (normothermic ischemic kidneys). Following this period of ischemia the kidney was removed with its vascular pedicle stump and ureter intact and perfused as described above. Experimental Protocol Kidneys subjected to 45 min of normothermic ischemia were perfused ex vivo for 120 min with one of three solutions. The first (Krebs, n = 6) was a Krebs-bicarbonate solution containing 7.5 g/dl of bovine serum albumin, 1 mg/ml glucose, 0.5 mg/ml creatinine, a mixture of amino acids, and trace amounts of [3H]inulin (glomerular marker). In the second solution (PCr + Krebs, n = 6), 3 mM phosphocreatine was added to the Krebs bicarbonate solution. The third solution was a modification of the “UW” solutions (PCr + R-SOL, n = 6) and consisted of 100 mM K+ lactobionate, 2.5 mM NaKH,PO,, 30 n&f raffinose, 5 mA4 MgSO,, 3 mM glutathione, 3 mM phosphocreatine, and trace amounts of [3H]inulin (osmolarity, 300-310 mOsm/liter). After an initial 10 min equilibration period, urine and perfusate samples were collected at 10 min clearance intervals. Perfusate flow was recorded from a Brooks flow meter in series with the arterial cannula. Urine volumes (V) were determined gravimetrically by collecting the urine in preweighed vials. Aliquots of perfusate (P) and urine (U) were placed in scintillation fluid (Aquasol) and analyzed for 3H radioactivity. Sodium (Na+) was determined by flame spectrophotometry (Turner Model 510). Glomerular filtration (GFR, ml/min) for each timed interval was calculated from the clearance of [3H]inulin ([U/P]in X V). The fractional reabsorption of water was calculated using inulin as a marker (l-[P/ U-L,, UJin). The fractional reabsorption of Na(FR,,) was calculated using the formula (l-[U/PJNa/[U/P] in).
Data are expressed as means f SE. Paired and unimpaired Student t test was utilized wherever appropriate. P < 0.05 was considered significant. RESULTS The results seen in Figs. 1 and 2 are given for nonischemic control kidneys (CONTROL) and ischemic kidneys perfused with either Krebs, PCr Krebs, or PCr + R- Sol. As shown in Fig. 1, kidneys subjected to 45 min of normothermic ischemia and subsequently perfused with Krebs solution were oliguric and had very low initial GRF with poor recovery. Kidneys treated with both Krebs + PCr solution and PCr + R-Sol had higher initial GFR values. The improvement in GFR values was sustained, reaching up to 80% of control values. Ischemic kidneys reperfused with phosphocreatine solutions showed high flow rates which where significantly improved over control values. These high flow rates were sustained through of 120 min of perfusion. In addition, urine flow rates were significantly enhanced with both phosphocreatine-containing solutions. The PCr + R-Sol perfused kidneys exhibited the highest urine flow rate, attaining values of up to 3 times control. The fractional reabsorption of sodium in the two PCr containing solutions were significantly improved over Krebs solution alone and closely approximated control values (see Fig. 2). In contrast fractional reabsorption of water differed in the PCr-KREBS solution compared with the PCr + R-Sol. The PCr + Krebs solution had slightly higher values for fractional reabsorption of water. DISCUSSION In general, experimental approaches to amelioration of renal ischemic injury have focused on the prevention or attenuation of intracellular swelling, prevention of
CONTROL KREBS PCr-KREBS PCr+R-SOL
FRN,
FR yo
FIG. 2. Effects of reperfusion of kidneys after 45 min of normothermic ischemia. Kidneys were subjected to 45 min of warm ischemia, then reperfused with solutions of Krebs, phosphocreatine added to Krebs (PCr-Krebs), and phosphocreatine added to a modified “WV” solution (PCr + R-sol) (see Methods for details). Fractional excretion of sodium (FR,,) and fractional excretion of water (FRH,O) were measured and the mean -C SE (n = 6 kidneys) was determined and compared to values obtained from nonischemic kidneys perfused with Krebs (control); +P c 0.05 compared to Krebs.
RODRIGUEZ
ET AL.: PHOSPHOCREATINE
IN RENAL
273
ISCHEMIA
Ca++ XANTHINE DEHYROGENASE (Ca++ PROTEASE)
k INOSINE
AMP HYPOXANTHINE
P PCr Cr
A N I E
REPERFUSION
i)
FIG. 3. As ischemic time increases ATP is progressively phosphorylated to AMP; once AMP is dephosphorylated to adenosine, adenosine leaks out of the cell. Adenosine is then metabolized to inosine and hypoxanthine, which in the presence of zanthine oxidase will react with reoerfused oxygen _- to create oxygen free radicals. In addition, the cell membrane becomes incompetent due to electrolyte pump failure permitting the influence of both sodium and calcium ions.
injury mediated through oxygen free radicals and removal (scavenging) of such agents, and the use of exogenous substrates to maintain the level of high energy phosphometabolites and total adenine nucleotide content in the cell. These three approaches address a continuum of changes shown diagramatically in Fig. 3. The increased tendency for cell swelling after reperfusion constitutes the basis for the use of perfusates during ischemia which mimic the “intracellular milieu.” One of the most efficacious has been the “UW solution.” This is a slightly hypoosmolar (300 to 310 mOsm) electrolyte solution containing raffinose (30 m&f) and lactobionate (100 mM). Raffinose is an impermeable trisaccharide and lactobionate is an impermeable disaccharide anion. Both raffinose and lactobionate have been proven to be much more effective impermeants than other substrates such as sodium citrate. This solution, while beneficial in organ preservation, does not address the issue of energy depletion (ATP dephosphorylation) or the leakage of adenosine from the cytosol. One approach has been to give high concentrations of PO, to inhibit adenosine deaminase, as well as providing high levels of adenosine in the perfusate to facilitate ATP synthesis [8]. The main problem with these agents is that they are rapidly degraded by cell membrane enzymes. Therefore, increasing the concentration of adenosine extracellularly may not result in osmotic equilibration across the cell membrane. Rather, there will be increased concentration of inosine and hypoxantine, its metabolic products. The mechanisms of “free radical” damage created by the increasing concentrations of inosine and hypoxantine are well described. Needless to say there have been many attempts at ameliorating renal ischemic injury by use of free oxygen radicals scavengers. These studies, however, have met with mixed results.
Phosphocreatine is the major high energy phosphometabolite of a number of organs including the brain, skeletal muscle, heart, and skin [9]. Its main theoretic advantages are its ability to penetrate the cell membrane and the potential for the high energy phosphate group to be translocated by cell membrane kinases, normally found in mitrochondria [lo]. Thus providing exogenous phosphocreatine to ischemic tissues may provide a readily available source of high energy phosphate to be used in the resynesthesia of ATP. Since ATP is not freely interchangeable but is compartmentalized to different parts of the cell as membrane-bound ATP-kinase complexes, phosphocreatine functions as the “energy currency” [ 111 of the cell delivering needed high energy phosphates to different membrane-bound ATP kinase complexes. Furthermore, the creatine base serves to buffer the production of hydrogen ions that occurs during ischemia. In the cytoplasm, the ATP pool is preserved at the expense of phosphocreatine. The maintenance of a pool of phosphorylated adenine nucleotides will effectively prevent leakage of adenosine. This will decrease the pool of reagents necessary for free oxygen radical production (inosine and hypoxanthine). Although the efficacy of phosphocreatine added to solutions perfusing skin flap [7] or heart [12] models have been reported, the role of phosphocreatine in the kidney has not been well delineated. Previous 31PNMR spectroscopy studies have detected low levels of phosphocreatine in the kidney [13]. In addition, whole renal tissue levels of creatine kinases have been reported as low, composed exclusively of the “brain” isozyme but not the “muscle type” [lo]. These observations no doubt are responsible for the relative paucity of studies addressing the role of phosphocreatine in recovery of postischemic renal injury.
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More recently, enzymatic assays have confirmed the presence of significant levels of phosphocreatine in the kidneys [14]. Further, this phosphocreatine pool responds predictably to ischemic insult. Phosphocreatine concentration in the kidney appears to vary, ranging from about 2 mmole/kg (dry weight) in the proximal straight tubule, thick ascending Loop of Henle, and papilla to about 10 mmole/kg in the distal convoluted tubule. The level of phosphocreatine in the distal convoluted tubule is comparable to that in brain. In comparison, ATP levels in proximal tubule segments are about 10 mmole/kg (dry weight) and 16 to 17 mmole/kg (dry weight) in the distal tubule segments. Renal creatine kinase activity varies regionally loo-fold. Measured levels were highest in the distal convoluted tubule and lowest in the proximal convoluted tubule. In the thick ascending Loop of Henle as well as in the distal convoluted tubule, phosphocreatine is thought to be consumed at the expense of ATP 1141. If indeed, phosphocreatine does play a role in energy metabolism in the kidney, exogenously administered phosphocreatine after a period of ischemia should help in the functional recovery of the kidney. In this study, we compared a solution which closely approximates the “UW” solution in that it contains the impermeant anions lactobionate and raffinose, the free radical scavenger, glutathione, and the energy substrate, phosphocreatine. This solution was compared both to a standard electrolyte solution (Krebs) as well as Krebs containing phosphocreatine. If prevention of intracellular swelling is the main determinant in ameliorating renal ischemic injury, one would expect to observe a significant difference between ischemic kidneys reperfused with the PCr + R-Sol solution and those reperfused with PCr + Krebs solution. No such differences were observed. Previous studies utilizing ATP/magnesium chloride in the reperfusion of ischemic kidneys demonstrated a beneficial effect on renal function but with progressive diuresis [4]. This was also observed in our PCr-treated ischemic kidneys. The increase in urine flow rate was more marked in the PCr + R solution and was associated with a slightly decreased (but not statistically significant) fractional reabsorption of water. The increase in urine flow rate and the decreased fractional reabsorption of water in the PCr + R solution may be due to the presence of the impermeant anions raffinose and lactobionate which when present in the tubular urine may diminish the passive reabsorption of water. While this effect might be beneficial in cold storage preservation of kidneys, in this study it did not result in any significant difference in amelioration of renal ischemic injury as compared to the Krebs + PCr solution. The fractional resorption of sodium, however, was improved for both the PCr + Krebs solution and the PCr + R solutions. This is an energy-requiring step and suggests that intracellular energy stores were replenished.
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The findings of this study establish that phosphocreatine has beneficial effects on the recovery of renal function following ischemia. The mechanism of this effect appears to be independent of the electrolyte composition of the solution. Use of an electrolyte solution which was designed to diminish intracellular swelling did not add any substantial benefit to that obtained by phosphocreatine in combination with a standard electrolyte solution. It suggests that phosphocreatine ameliorates renal ischemic injury by repletion of phosphometabolite balance and may prevent adenosine leakage which leads to “free radical” generation. ACKNOWLEDGMENT This work was supported in part by an OHSE Grant from the Department of Surgery, Yale University School of Medicine.
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