Transplantation of a solid organ from a donor has emerged as a treatment option for many diseases. Both the interruption and subsequent restoration of blood flow are often the cause of tissue injury in any transplanted organ. Despite the progress in surgical techniques and preservation conditions1, every transplantation starts with an inevitable insult of the graft: the ischemia-reperfusion syndrome. Ischemia-reperfusion injury may be considered an inflammatory and vasomotor phenomenon. In renal transplant, delayed graft function is a relatively common complication of this syndrome, occurring between 20% and 60% of cadaveric kidney recipients2. It makes the diagnosis of rejection more difficult, prolongs hospital stay, renders allografts more immunogenic and prone to rejection, and ultimately reduces short- and long-term graft survival rates3,4,5. Thus, the transplant community has focused increased attention on identifying drugs that stabilize or reverse ischemia-reperfusion injury.
The primary events that can be considered responsible for the cascade of metabolic, functional and structural alterations that develop after ischemia are energy imbalance and alterations of cellular homeostasis6. Moreover, the reperfusion of previously ischemic tissue potentiates influx of calcium, release of intracellular enzymes, breakdown of phospholipids and disruption of cell membrane integrity, which either alone or in combination results in ultimate cell death6. Current evidence leads to at least four major hypotheses concerning the mediators of ischemia-reperfusion injury: calcium overloading, generation of oxygen-derived free radicals, degradation of phospholipids, and accumulation of long-chain acylcarnitines6,7,8,9.
Interventions with calcium entry blockers8,10, free radical scavengers11,12,13, drugs that affect phospholipid metabolism and activity14,15 or up-regulate 
-oxidation of long-chain fatty acids16 have resulted in a significant but incomplete degree of protection in different experimental models of heart and renal ischemia-reperfusion injury. Only very few studies have addressed this issue in experimental renal transplantation.
In this regard, carnitine and propionyl-L-carnitine have been reported to inhibit free radicals generation17,18, protect against impairment of fatty acid -oxidation in mitochondria19, inhibit platelet-activating factor20 and preserves cell membrane integrity21,22 and induce vasodilation23. Thus, these agents could target simultaneously more than one pathogenetic factor of ischemia-reperfusion injury eventually providing better therapeutic potential than that afforded by compounds so far explored in these experimental models.
Carnitine is an essential cofactor required for the translocation of activated long-chain fatty acids from extramitochondrial coenzyme A (CoA) into the inner mitochondrial matrix and then to intramitochondrial CoA, a process that requires carnitine and carnitine acyltransferase24. This transport is essential to allow mitochondrial -oxidation of long-chain fatty acid24 and thus to provide energy supply to the cells. Therefore, carnitine is an important factor in regulating substrate flux and energy balance across cell membranes, which might become critical in the context of ischemia-reperfusion, possibly preventing cell injury. Propionyl-L-carnitine, a short-chain acyl derivative of L-carnitine, has the potential not only of restoring tissue carnitine stores, but also to replenish key mitochondrial tricarboxylic acid intermediates as documented by studies in skeletal muscle mitochondria25 and in ischemic heart tissues26,27
The beneficial effects of carnitine and propionyl-L-carnitine have been documented in animal models of heart ischemia–reperfusion injury26,28,29,30,31. Whether these compounds are also of value in preventing ischemia-reperfusion injury in the kidney has not been examined so far.
The present study was designed with the following aims: (1) to investigate whether carnitine and propionyl-L-carnitine prevented renal function deterioration and tubular injury induced by ischemia/reperfusion in a rat model of isolated perfused kidney (IPK); (2) to evaluate whether the treatments may afford protection even when renal injury is already established; (3) to examine the possible mechanism(s) involved in the beneficial effect, if any, of propionyl-L-carnitine in this model; and (4) to explore whether addition of propionyl-L-carnitine to Belzer UW solution during cold storage of donor kidney prevents ischemia-reperfusion injury and facilitates immediate graft function in an experimental model of syngeneic rat kidney transplantation.
METHODS
Animals
Adult male Sprague-Dawley rats of 300 to 350 g body weight (Charles River Italia Spa, Calco, Italy) were used in IPK studies. Inbred adult male Lewis rats (RT1l, Charles River) were used as donors and recipients in renal transplantation studies.
Animal care and treatment were conducted in accordance with institutional guidelines in compliance with national (D.L. n.116, G.U. suppl. 40, February 18, 1992, Circolare n.8, G.U. July 14, 1994) and international laws and policies (European Economic Community Council Directive 86/609, OJL 358, December 1987; "Guide for the Care and Use of Laboratory Animals", U.S. National Research Council, 1996). All animals were allowed free access to standard rat chow and tap water.
Isolated kidney perfusion procedures and apparatus
The perfusion technique used in these experiments has been previously described in detail32. The kidney was perfused in situ in a recirculating system with a medium held at 37°C by a constant Haake D1 temperature circulator system (Haake, Berlin, Germany) and gassed with a mixture of 95% O2-5%CO2 through a hallow-fiber membrane oxygenator. The perfusate was delivered to the renal artery cannula through a peristaltic pump, an in-line 8-
m pore-size filter (Sartorius, Gottingen, Germany), and a glass bubble trap.
The perfusate consisted of Krebs-Henseleit bicarbonate buffer containing 3.5 g/dL of Ficoll 70 (Pharmacia Fine Chemical, Uppsala, Sweden), 1 g/dL of bovine serum albumin (BSA; Pentex BSA Fraction V; Bayer, Kankakee, IL, USA), 200 mg/dL of glucose, 36 mg/dL of urea, 50 mg/dL of creatinine, and a mixture of amino acids as described32. The total volume of the perfusate in the system was 250 mL.
Urine flow rate was determined gravimetrically. Perfusate and urinary concentration of creatinine were measured by Jaffè assay33 and the glomerular filtration rate (GFR) calculated as creatinine clearance according to standard formula34. Renal perfusion flow (RPF) was determined volumetrically. The perfusion pressure was kept constant at 100 mm Hg throughout the experiments. Therefore, changes in RPF reflected changes in renal vascular resistance. For tissue collection a three-way stop-cock was incorporated into the circuit near to the arterial cannula to allow perfusion with the fixative solution at the same pressure applied during the functional study, for an additional 10 to 15 minutes. The fixative solution contained 4% p-formaldehyde, (plus 5% sucrose), and butylated-hydroxy-toluene (50 mol/L) and 1 mmol/L ethylenediaminetetraacetic acid (EDTA). Kidney specimens were collected and processed for light microscopy analysis.
Kidney transplantation
Kidney transplantation was performed as described previously35. The donor kidney was flushed with Belzer UW solution containing 1000 U/mL heparin and added or not with propionyl-L-carnitine, according to the experimental group and placed in an iced Belzer UW solution for four hours with or without propionyl-L-carnitine (cold ischemia) until transplant. After ischemia time had elapsed, recipient rats underwent removal of the native left kidney. Donor kidneys grafts were then washed with saline solution and transplanted. An anastomosis was created between the donor and recipient renal artery as well as renal vein with end-to-end anastomosis. Vascular clamps were released after 30 minutes (warm ischemia). Donor and recipient ureters were attached end-to-end. The native right kidney was then removed.
Histological examination
Kidney specimens from IPK and syngeneic transplanted animals were fixed in Dubosq-Brazil and dehydrated in alcohol. After paraffin embedding, 3-m sections of the blocks were cut and stained with periodic acid-Schiff (PAS), Masson's trichrome, and with hematoxylin eosin (H&E). The histological evaluation was performed in a blinded fashion on representative sections of cortex and outer medullary regions. In control IPK preparations only a mild and focal vacuolization of proximal tubular epithelial cells was detected in the cortex. Histologic lesions in the medulla consisted of cell swelling and cytoplasmic disruption progressing to cell necrosis in the thick ascending limbs, mainly in the inner stripe region, and vacuolization, loss of brush border, and luminal debris in proximal tubules. Structural changes were assessed on H&E stained sections using a semiquantitative scale with increasing severity of damage in 
20 randomly selected fields (at least 15 to 20 fields) for each section and the fields were assigned individual scores. For thick ascending limbs, lesions were graded as follows: 0 = no abnormalities; 1 = degenerative changes, with only occasional necrotic cells; 2 = lesions of moderate severity, with necrosis involving approximately 
50% profiles; 3 = extensive necrosis. For proximal tubules, lesions were graded as follows: 0 = no abnormalities; 1 = focal vacuolization and loss of brush borders; 2 = lesions of moderate severity with blebbing and loss of individual cells; 3 = severe lesions.
In syngeneic kidney graft tissues, tubular damage (defined as cell swelling, vacuolization, necrosis and loss of brush border) was assessed and graded according to a semiquantitative scale (0 to 3)36. No or very mild ischemic lesions were found in the glomeruli36.
The degree of leukocyte infiltration in glomerular, perivascular and interstitial areas was determined by indirect immunofluorescence. Mouse monoclonal antibodies were used for the detection of the following antigens: (1) ED1 antigen, present in the rat monocytes and macrophages (Chemicon, Temecula, CA, USA); (2) a rat major histocompatibility complex (MHC) class II antigen monomorphic determinant (OX6; Serotec, Oxford, UK); (3) rat CD8 cell surface glycoprotein expressed by T cytotoxic suppressor cells (OX8; Serotec). A mouse anti-rat granulocyte monoclonal antibody (clone MOM/3F12/F2; Serotec) was used to stain infiltrating neutrophils. For these analyses tissue fragments were frozen in liquid nitrogen, sections (3 m thick) cut using a Mikrom 500 O cryostat (Walldorf, Germany) and fixed in acetone. Tissue sections were blocked with phosphate-buffered saline (PBS)/1% BSA, incubated overnight at 4°C with the primary antibody (ED1, 14 mg/mL; OX6, 5 g/mL; OX8, 1:100, MOM/3F12/F2, 1:10), washed with PBS, and then incubated with Cy3-conjugated donkey anti-mouse IgG antibodies (affinity-purified, absorbed with rat IgG, 5 g/mL in PBS; Jackson ImmunoResearch, West Grove, PA, USA) for one hour at room temperature. For each marker, the number of cells was counted in at least ten randomly selected high-power microscope fields (400) for each animal.
Adenine nucleotides measurement
The freeze-clamped tissue was stored at -80°C until analysis. Tissue was homogenized in HClO4 (14%, vol/vol) and subsequent adenine nucleotides analyses of the neutralized perchloric extracts were carried out with a high-pressure liquid chromatography (HPLC) method, as previously described37.
Immunocytochemistry of 4-hydroxynonenal protein adduct in IPK
Lipid peroxidation in IPK was localized by a specific anti-4-hydroxynonenal-lysine (4-HNE-lysine) antibody (NA59, kindly provided by Dr. Witzum, The Scripps Research Institute, La Jolla, CA, USA)38. After perfusion fixation, biopsy slices (2 mm thick) were cut perpendicularly to the major axis of the kidney. Three-m paraffin sections from renal tissue were processed for immunohistochemistry using an avidin-biotin horseradish peroxidase complex technique (ABC method, ABC-Elite; Vector Laboratories, Burlingame, CA, USA)39. Briefly, the sections were dewaxed, rehydrated and incubated for 30 minutes with 0.3% H2O2 in methanol to quench endogenous peroxidase. Tissue was permeabilized in 0.1% Triton X-100 in PBS 0.01 mol/L, pH 7.2, for 30 minutes and aspecificities were blocked by a 30-minute incubation with non-immune horse serum. All the above steps were carried out at room temperature. Slides were then incubated overnight at 4°C in moist chamber with the primary anti-4-HNE-lysine antibody (1:500) in PBS/1% BSA, followed by the biotinylated horse-anti-mouse IgG, ABC solution (Vector Laboratories) and developed with diaminobenzidine/nickel. The sections were then counterstained with Harris hematoxylin (Biooptica, Milan, Italy). Negative controls were obtained by omitting the primary antibody on a second section present on all the slides. The slides were examined under light microscope by two pathologists blind to the nature of the experiment.
Multiple sections from each IPK were examined. Each section was scored for intensity of immunostaining (absent, faint, moderate, intense: 0 through 3). At least 8 to 10 fields per section were examined. The mean values for each section and for each IPK were determined.
Isolated perfused kidney study design
To assess whether propionyl-L-carnitine may preserve renal function in conditions of ischemic insult to the kidney, we first performed ex vivo experiments using a model of ischemia/reperfusion injury in isolated rat kidney perfused in a circulating system with an artificial cell-free medium Figure 1a. After surgery and a 20 to 25 minute equilibration period, a ten-minute baseline urine collection and a perfusate sample were obtained (control clearance). Thereafter, kidneys were exposed to ischemia by markedly reducing perfusion pressure (10 mm Hg) for 20 minutes. Perfusion pressure was then restored to normal (100 mm Hg) and additional nine consecutive ten-minute clearance periods were performed. Three experimental groups of IPKs were considered: group 1 (N = 10), was infused with vehicle (saline) directly into the renal artery; group 2 (N = 10) was exposed to propionyl-L-carnitine (3.6 mg/mL, final concentration) infused at the rate of 0.4 mL/min. The concentration of propionyl-L-carnitine was chosen according to preliminary dose-response safety experiments in normal IPK preparations (data not shown). For comparison an additional group (group 3, N = 3), was infused with L-carnitine (3.6 mg/mL f.c.; Sigma Tau, Rome, Italy), at the same rate of 0.4 mL/min. As a control group (group 4, N = 6), kidneys infused with vehicle but not undergoing ischemia/reperfusion injury were studied. All treatments started at the beginning of the control clearance period and continued for the two-hour perfusion period. Perfusion with vehicle, propionyl-L-carnitine, or L-carnitine was performed using an appropriate infusion pump connected with a catheter to the circuit very proximally to the renal artery. For each collection period urine and venous effluent samples were collected for measurement of urine output, glomerular filtration rate (GFR), and renal perfusate flow (RPF).
Figure 1.
Experimental protocol. (A) Isolated perfused rat kidney (IPK). After equilibration (time 0) and a 10 min. baseline period, kidneys were exposed to ischemia by reducing perfusion pressure to 10 mm Hg. Perfusion pressure was then restored to normal (100 mm Hg) and additional 9 consecutive 10-minute clearance periods were performed. Seven experimental groups were studied: group 1, IPK undergoing 20 minutes of ischemia and infused with vehicle; group 2, IPK undergoing 20 minutes of ischemia and infused with propionyl-L-carnitine from time 0; group 3, IPK undergoing 20 minutes ischemia and infused with L-carnitine from time 0; group 4, control IPK not undergoing ischemia; group 5, IPK undergoing 40 minutes of ischemia and infused with vehicle; group 6, IPK undergoing 40 minutes of ischemia and infused with propionyl-L-carnitine from time 0; group 7, IPK undergoing 20 minutes of ischemia and infused with propionyl-L-carnitine starting immediately after reperfusion (not shown in figure). (B) Syngeneic kidney transplant. Donor kidneys were transplanted in the syngeneic nephrectomized recipients after 4 hours of cold ischemia time in Belzer UW solution. Five experimental groups were studied: group 8, donor kidney transplanted after a 4-hour cold ischemia in Belzer plus vehicle; group 9, donor kidney transplanted after a 4-hour cold ischemia in Belzer plus propionyl-L-carnitine; group 10, donor kidney transplanted, no cold ischemia; group 11 (not shown), donor kidney transplanted after 4 hours of cold ischemia in Belzer plus vehicle, recipients sacrificed 16 hours post-transplantation; group 12, (not shown), donor kidney transplanted after 4 hours of cold ischemia in Belzer plus propionyl-L-carnitine, recipients sacrificed 16 hours after transplantation.
Full figure and legend (61K)The same experimental protocol was repeated to assess the effect of propionyl-L-carnitine on renal function in conditions of more severe ischemic insult to the kidneys, which more closely mimics the peritransplant state that occurs when the ischemic period is prolonged up to 40 minutes, again by reducing perfusion pressure to the IPK. Thus, kidneys undergoing ischemia/reperfusion were exposed to vehicle (saline, group 5, N = 3) or propionyl-L-carnitine (3.6 mg/mL f.c.; infusion rate 0.4 mL/min, group 6, N = 3) with the infusion starting at the beginning of the experimental period (pre-ischemia/reperfusion) and continued during the two-hour perfusion period. Urine output, GFR, and RPF were monitored Figure 1a.
In additional experiments the therapeutic effect of propionyl-L-carnitine was also investigated. Therefore, after a ten-minute control clearance period and 20-minute ischemia, kidneys were infused with propionyl-L-carnitine (3.6 mg/mL f.c., 0.4 mL/min, group 7, N = 6), starting immediately after reperfusion was restored and then continuing until the end of the perfusion period. Renal function was assessed as above.
To examine whether exposure to propionyl-L-carnitine affected tubular structure, some of the experiments analyzed renal tissue specimens by light microscopy at the end of the perfusion period. Since minor differences were found in IPK exposed to 20 or 40 minutes of ischemia at histological examination, score values of the two sets of experiments were pooled for statistical analysis. Control non-ischemic kidneys (N = 3), ischemic kidneys (N = 5), ischemic kidneys exposed to propionyl-L-carnitine (N = 6) were considered.
In additional experiments, to evaluate whether the potential protective effect of propionyl-L-carnitine occurred by preserving renal cell viability, we measured the time course of lactate dehydrogenase (LDH) release (by an automatic analyzer) in the renal vein effluent in control IPK preparations, in those exposed to 20 minutes of ischemia, and those in which propionyl-L-carnitine had been infused before ischemia was established. Measurements were performed in perfusate samples collected in subgroups of the above-mentioned set of experiments.
Moreover, whether the possible protective effect of propionyl-L-carnitine on ischemia-reperfusion injury was related to inhibition of lipid peroxidation process was investigated using two different methodological approaches: (1) by immunohistochemical detection of 4-hydroxynonenal, a major aldehydic product of lipid peroxidation40, believed to be largely responsible for cytopathological effects observed during oxidative stress41, on the renal tissue sections obtained for morphological analysis; and (2) by evaluating the concentration of MDA, using the thiobarbituric acid method42, in the venous effluent collected at various time points during the 120-minute perfusion period.
To examine whether propionyl-L-carnitine improved the energy metabolism of the ischemic kidney, additional experiments were performed with kidneys undergoing 20 minutes of ischemia/reperfusion exposed to vehicle (saline, N = 4) or propionyl-L-carnitine (N = 4). At the end of the 120-minute perfusion period the kidneys were freeze-clamped in liquid nitrogen for the evaluation of high energy phosphates.
Kidney transplant study design
To evaluate whether propionyl-L-carnitine also may exert a protective effect in vivo, experiments were performed in a model of syngeneic rat kidney transplantation, and the impact of the treatment on renal function preservation during ischemia/reperfusion injury due to cold kidney storage and revascularization was studied Figure 1b. The absence of alloreactivity in this model allowed us to isolate the effect of cold ischemia from immunological factors. Donor kidneys were transplanted in the syngeneic nephrectomized recipients after four hours of cold ischemia time in Belzer UW solution (ViaSpan; DuPont Pharma, Firenze Italy; group 8, N = 7) or the same solution containing propionyl-L-carnitine (1.2 mg/mL, group 9, N = 7). This concentration of propionyl-L-carnitine was chosen based on preliminary experiments showing toxicity soon after transplantation when higher doses (3.6 and 1.8 mg/mL) of the compound were used. Nephrectomized animals receiving a syngeneic graft not subjected to cold ischemia (non-ischemic grafts) were used as controls (group 10, N = 3). Warm ischemia time during surgical procedures was approximately 30 minutes. At 16 hours, 24 hours, and on days 2, 5, 7 and 30 post-transplant, renal function was assayed by measuring plasma creatinine concentration, by a Reflotron creatinine test (Boehringer Mannheim, Mannheim, Germany), on whole blood collected from the tail vein of anesthetized animals.
Two additional groups of rat recipients of a syngeneic kidney exposed to four hours of cold storage in Belzer UW solution alone (group 11, N = 4) or added with propionyl-L-carnitine (1.2 mg /ml, group 12, N = 4) were studied to specifically investigate the effect of propionyl-L-carnitine on graft histology and inflammatory cell infiltrate. The animals were sacrificed 16 hours after transplantation, the kidney grafts removed, and processed for structural examination and immunohistochemical analysis of polymorphonuclear cell, CD8 T lymphocyte, macrophage, and MHC class II positive cell infiltrate.
Statistical analysis
Results are expressed as mean 
SD or SE as specified in the Figure legends. Data were analyzed using two-way, one-way analysis of variance (ANOVA), or Kruskal-Wallis as appropriate. The significance level of difference between individual group means, subjected to the two-way ANOVA, was established using the Tukey-Cicchetti test for multiple comparisons43. Statistical significance was defined as P < 0.05.
RESULTS
Effect of propionyl-L-carnitine on renal function deterioration in a model of ischemia-reperfusion in the IPK
In control non-ischemic kidneys perfused with vehicle, urine output tended to increase progressively over the two-hour perfusion period (baseline, 86 21; 60 min, 104 15; 120 min, 118 34 L/min). In ischemic kidneys exposed to vehicle urine flow markedly declined at the beginning of the ischemia period when perfusion pressure was intentionally lowered to allow a minimal perfusion flow. When reperfusion was established, diuresis progressively increased reaching pre-ischemia values 20 minutes later (pre-ischemia, 107 32 L/min vs. 20 min post-ischemia, 113 27 L/min). This was maintained for the subsequent 30 minutes and then diuresis spontaneously declined (end of perfusion, 104 36 L/min). In kidneys exposed to propionyl-L-carnitine before ischemia, urine output after reperfusion promptly recovered and 20 minutes later was numerically higher than pre-ischemia values (pre-ischemia, 105 11 L/min vs. 20 min post-ischemia, 128 13 L/min). Thereafter, urine output remained quite constant up to the end of the two-hour observation period (127 26 L/min). A similar profile of urine output was also found with L-carnitine pre-treatment (pre-ischemia, 90 31 L/min; end of perfusion: 125 16 L/min). No significant difference among groups as far as urine output was documented at any time of the perfusion period.
In control non-ischemic kidneys perfused with vehicle renal function slightly declined during the two-hour study period, according to the characteristics of this preparation32 (baseline GFR, 1.09 0.14; 60 min GFR, 1.03 0.15; 120 min GFR, 0.78 0.14 mL/min). As shown in Figure 2a, in ischemic kidneys treated with vehicle, the GFR that was not measurable during the ischemia period was restored after establishing reperfusion, but the values were significantly lower than pre-ischemia GFR (0.47 0.12 vs. 1.06 0.19 mL/min, P < 0.01). After a mild tendency to increase during the subsequent 20 minutes of perfusion, a further progressive decline of GFR was documented (end of perfusion, 0.48 0.16 mL/min). In kidneys exposed to propionyl-L-carnitine before ischemia, GFR recovered more promptly than in vehicle-treated kidneys, as shown by a significantly higher mean value at the end of the first ten-minutes after reperfusion (0.74 0.17 mL/min, P < 0.01 vs. vehicle-treated kidneys). GFR progressively increased during the subsequent 30 minutes of perfusion, with a slight decrease thereafter, but the values were always significantly higher than in vehicle-treated kidneys for the entire observation period (GFR at end of perfusion, 0.78 0.17 mL/min, P < 0.01 vs. vehicle). Also, pre-treatment with L-carnitine largely prevented the decline in GFR after reperfusion, although a tendency for mean values to decline with time was observed (pre-ischemia, 0.97 0.12; 20 min of reperfusion, 0.72 0.08; end of perfusion, 0.64 0.07 mL/min).
Figure 2.
Effect of propionyl-L-carnitine on changes in glomerular filtration rate (GFR, A) and renal perfusate flow (RPF, B) induced by 20 minutes of ischemia in an isolated perfused rat kidney preparation. Vehicle (
; N = 10), propionyl-L-carnitine (
; N = 10) or L-carnitine (
; N = 10) was infused into the renal artery starting before or immediately after the ischemic period. Values are mean SD. *P < 0.01 vs. propionyl-L-carnitine and baseline; P < 0.05 vs. baseline.
As for RPF, a minor progressive decline of values with time was found in control non-ischemic kidneys exposed to vehicle (baseline RPF, 27.3 1.5; 60 min RPF, 26.6 1.2; 120 min RPF, 25.5 1.0 mL/min). In ischemic kidneys perfused with vehicle RPF declined from 26 1 mL/min at baseline to 5.7 0.4 mL/min at the beginning of ischemia period Figure 2b. When normal perfusion was re-established, RPF returned toward but did not reach pre-ischemia values (21 2 mL/min, P < 0.01 vs. pre-ischemia). Thereafter, RPF remained at values significantly lower than baseline up to the end of the two-hour perfusion (24 1 mL/min). In kidneys exposed to propionyl-L-carnitine starting before ischemia, RPF was comparable to that in vehicle-treated kidneys before (27 1 mL/min) and during the ischemia period (5.3 0.5 mL/min). After reperfusion, RPF recovered faster and values were numerically but not significantly lower than pre-ischemia for most of the remaining perfusion period (end of perfusion 25 1 mL/min). A similar RPF profile was observed in kidneys exposed to L-carnitine (pre-ischemia, 26 0; end of perfusion, 24 1 mL/min).
Table 1 shows the effect of vehicle or propionyl-L-carnitine on renal function parameters before and after a more severe (40 min) ischemia period. In vehicle-treated kidneys, GFR increased after reperfusion but largely remained below pre-ischemia values (P < 0.01 vs. baseline at all points considered). In kidneys exposed to propionyl-L-carnitine GFR recovered more promptly than in vehicle-treated kidneys (P < 0.05 at 60 min), but values were still significantly lower than pre-ischemia (P < 0.05). As for RPF, in vehicle-treated kidneys RPF tended to increase after reperfusion, but remained significantly lower than pre-ischemic values at all time points considered Table 1. In kidneys exposed to propionyl-L-carnitine RPF recovered faster and at 90 minutes of perfusion was only numerically lower than pre-ischemia values.
Table 1 - Effect of propionyl-L-carnitine on renal function parameters in a model of ischemia (40 min)/reperfusion in isolated perfused kidneys.
Full table Therapeutic effect of propionyl-L-carnitine on renal function deterioration in a model of ischemia-reperfusion in IPK
Infusion of propionyl-L-carnitine starting at the beginning of reperfusion resulted in marked increase of GFR, despite the recovery of this parameter was less prompt than in kidneys pre-exposed to the compound (pre-ischemia, 1.10 0.06 mL/min; 10 min post-ischemia, 0.43 0.05 mL/min). Thereafter, GFR values were comparable to those achieved in kidneys exposed to propionyl-L-carnitine from the beginning of the perfusion (end of perfusion, 0.80 0.07 mL/min vs. 0.78 0.17 mL/min) and were significantly higher than in vehicle-treated kidneys (P < 0.01). Similar results were obtained for RPF, whose values increased with time to the same extent independently of whether propionyl-L-carnitine infusion began before or after ischemia (end of perfusion, 25 1 mL/min vs. 26 2 mL/min).
Effect of propionyl-L-carnitine on biochemical and histological markers of tissue damage in the model of ischemia-reperfusion in IPK
In control kidneys a mild, progressive increase with time in the amount of LDH released in the perfusate was found Table 2. In those kidneys exposed to 20 minutes of ischemia, reperfusion was associated with a marked percent increase of LDH release as compared to baseline pre-ischemia values, with a further progressive increase through the end of the observation period. Percent LDH changes was significantly higher than that reported in control kidneys. Pre-exposure of ischemic kidneys to propionyl-L-carnitine largely reduced, but not normalized the percent of LDH released in the perfusate after reperfusion as compared to baseline.
Table 2 - Effect of propionyl-L-carnitine on lactate dehydrogenase (LDH) release into the perfusate venous effluent in isolated perfused kidneys.
Full table Results of scoring evaluation of tubular damage are summarized in Figure 3. Sections obtained from control IPK showed degenerative changes in thick ascending limbs and proximal tubule segments located in the outer medulla Figure 4, a, b. Thick ascending limbs showed severe changes, typical of this model44,45, with marked swelling of the cytoplasm and cell detachment or necrosis. Lesions were more pronounced in kidneys exposed to 20 or 40 minutes ischemia, which in addition to severe damage of thick ascending limbs showed much more severe proximal tubular injury, including necrosis and cell debris in tubular lumen Figure 4, c, d. IPKs pre-exposed to propionyl-L-carnitine and undergoing ischemia/reperfusion had significantly Figure 3 less severe post-ischemic tubular lesions as compared to untreated ischemic kidneys. The protective effect of propionyl-L-carnitine was remarkable both in the thick ascending limbs and, to a lesser extent, in the proximal tubuli Figure 4, e, f.
Figure 3.
Tubular histological change score in control isolated perfused kidneys (IPKs; Control, N = 3), in kidney exposed to 20 or 40 minutes of ischemia and treated with vehicle (Isch/reper + vehicle, N = 5), or propionyl-L-carnitine (Isch/reper + propionyl-L-carn nitine, N = 6). Symbols are: () TAL, thick ascending limbs; (
) PCT, proximal convoluted tubules. Values are mean SD. *P < 0.05 vs. Isch/reper + vehicle, §P < 0.05 vs. control and Isch/reper + propionyl-L-carn.
Figure 4.
Histological appearance of fields of inner stripe (A, C, E) or outer stripe (B, D, F) of outer medulla of a control isolated perfused rat kidney (A, B), a kidney exposed to 20 minutes of ischemia/reperfusion (C, D), and a kidney infused with propionyl-L-carnitine and exposed to 20 minutes of ischemia/reperfusion (E, F) (H&E, 500).
Table 3 shows that the concentrations of energy compounds (ATP and ADP) and total energy charge [0.5 (ADP+ 2ATP)/(ATP+ADP+AMP)] were low in IPKs undergoing ischemia/reperfusion, as a consequence of ischemia-induced loss14,22,46. Pre-exposure to propionyl-L-carnitine had no effect on ATP, energy charge, adenine nucleotides and adenine content in the IPKs undergoing ischemia/reperfusion Table 3.
Table 3 - Adenine nucleotides, adenosine levels and energy charge in isolated perfused rat kidneys.
Full table To evaluate the extent of generation of lipid peroxidation products and their cell localization in the isolated kidneys after ischemia/reperfusion and the potential protective effect of propionyl-L-carnitine, the amount and the distribution of 4-HNE-modified proteins using a specific anti-4-HNE-lysine antibody were assessed38,47. Indeed, it has been shown that the formation of adducts between 4-HNE, a major aldehydic product of lipid peroxidation40,48,49,50, and the lysine residues of structural proteins, provides good evidence of local oxidative stress51. As shown in Figure 5 a and b, and Table 4, 4-HNE-lysine staining was mostly faint in cortical and medullary tubules of control kidneys perfused with saline, while glomeruli were mostly negative. By contrast, IPKs undergoing either 20 or 40 minutes of ischemia and exposed to vehicle showed moderate to intense 4-HNE-lysine staining in medullary tubules (Figure 5, c, d, and Table 4). In kidneys exposed to propionyl-L-carnitine before ischemia, 4-HNE-lysine staining was impressively reduced in tubules and glomeruli as compared to kidney exposed to vehicle, indicating that the drug protected renal tissue from oxidative damage Figure 5, e, f. No staining was found in the absence of primary antibody (not shown). MDA concentrations in the venous effluent at baseline were comparable in control IPK (11.6 2.9 nmol/mL, N = 4), in IPKs pre-exposed to vehicle and undergoing 20 minutes of ischemia (11.0 0.65, N = 4), and in IPKs pre-exposed to propionyl-L-carnitine and undergoing 20 minutes of ischemia (11.8 2.6, N = 4). In control IPKs MDA concentration in the venous effluent remained quite stable during the perfusion time. By contrast, in those kidneys exposed to 20 minutes of ischemia, reperfusion was associated with a marked increase of MDA release in the venous effluent as compared to baseline pre-ischemia values, with a maximal increase during the first 10 minutes of reperfusion (40 min, 41 10% increase over baseline, P < 0.05, vs. 40 min control IPK, 5 3% increase over baseline). Pre-exposure of ischemic kidneys to propionyl-L-carnitine completely abolished the increase of MDA in the venous effluent after reperfusion, as compared to baseline (40 min, -2.4 6% increase over baseline, P < 0.05, vs. IPKs pre-exposed to vehicle and undergoing 20 min ischemia).
Figure 5.
Representative photomicrograph of immunoperoxidase staining of outer medulla renal tissue with anti-4-HNE-lysine antibody. (A and B) Control IPK. (C and D) IPK undergoing 20 minutes of ischemia and exposed to vehicle. (E and F) IPK exposed to propionyl-L-carnitine before ischemia. Original magnifications are 100 (A, C, E) and 400 (B, D, F).
Table 4 - Immunohistochemical detection of 4-hydroxynonenal protein adducts in glomeruli and tubules of siolated perfused kidneys.
Full table Effect of propionyl-L-carnitine on ischemia-reperfusion-induced graft dysfunction in syngeneic kidney transplants
Figure 6 shows the time course of serum creatinine concentration in Lewis rats recipients of syngeneic kidney transplants pre-exposed to a four-hour cold ischemia period. In animals receiving untreated donor ischemic kidneys (group 8) renal dysfunction occurred early post-transplant, as documented by serum creatinine values significantly higher than in control rats transplanted with non-ischemic kidneys and studied at 16, 24 hours and at day 2 post-surgery. Thereafter renal function progressively improved with serum creatinine reaching normal values in most animals at day 5 of follow-up. Three out of seven rats, however, did not completely recover graft function, and one of them died at day 19 post-transplant. By contrast, in animals receiving kidneys treated with propionyl-L-carnitine during the pre-transplant four-hour ischemia period, serum creatinine at 16 and 24 hours post-surgery was significantly lower than values in animals transplanted with untreated ischemic kidneys (16 hr, P < 0.01; 24 hr, P < 0.05). Renal function completely recovered in these animals by day 5 post-transplant.
Figure 6.
Serum creatinine concentration in Lewis rat recipients of a syngeneic kidney pre-exposed to four hours of cold ischemia in Belzer UW solution (
, N = 7) or in Belzer solution added with propionyl-L-carnitine (1.2 mg/mL, , N = 7). Time 0 shows the serum creatinine concentration in the same animals before bilateral nephrectomy and transplantation. The gray bar represents the creatinine range in control recipients of a non-ischemic syngeneic kidney (N = 3). Values are mean SE. *P < 0.01, #P < 0.05 vs. ischemia untreated, P < 0.01 vs. control non-ischemic graft recipients.
Effect of propionyl-L-carnitine on ischemia-reperfusion-induced graft injury and cell infiltration in syngeneic kidney transplants
Histologic examination of sections from kidneys transplanted after four-hour cold ischemia and studied 16 hours after transplantation showed moderate tubular necrosis manifested by swollen, vacuolated proximal tubular cells with pyknotic nuclei and cell detachment (semiquantitative mean score, 1.1 0.3). Additional changes included tubular dilation, flattening of tubular epithelium and cell debris in tubular lumens36,52. Propionyl-L-carnitine partially reduced tubular damage (mean score, 0.8 0.2).
A detailed immunohistologic evaluation of intragraft leukocyte infiltration at 16 hours post-transplantation was also undertaken. Figure 7 summarizes the results of quantitative evaluation of neutrophils infiltration by immunohistochemical staining. In untreated ischemic kidneys a large number of granulocytes was found in the interstitium, and to a lesser extent in intra- and periglomerular areas as well as in perivascular areas. Addition of propionyl-L-carnitine to the cold storage solution significantly decreased graft granulocyte infiltration at 16 hours post-transplant (P < 0.05 vs. untreated ischemic grafts). In contrast to granulocytes, a low number of ED1+ monocytes/macrophages, MHC class II+ cells, and CD8+ cells was found in kidneys transplanted following four hours of ischemia, mainly in the interstitium Table 5. No difference was found in monocyte and T-cell infiltration between untreated and propionyl-L-carnitine treated grafts Table 5.
Figure 7.
Immunohistochemical staining for infiltrating granulocytes in the glomeruli (intra- and periglomerular), the perivascular area and the interstitium of syngeneic kidneys transplanted after four hours of cold ischemia in Belzer (UW) solution (
, N = 4) or in Belzer solution added to propionyl-L-carnitine (1.2 mg/mL, ,N = 4). All animals were studied 16 hours after transplantation. The number of cells was counted in at least 10 randomly selected high-power microscope fields (400) for each animal. Values are mean SE. #P < 0.05 vs. ischemia untreated.
Table 5 - Intragraft immunostaining of ED1+ cells (macrophages), OX6+ cells (MHC II+) and OX8+ cells (CD8+ T cells), in the interstitium of syngeneic transplanted kidneys.
Full table DISCUSSION
In an ex vivo model of ischemia-reperfusion injury in an isolated perfused rat kidney preparation, exposure of kidneys to propionyl-L-carnitine before the ischemic insult largely prevented renal function impairment that instead occurred after restoring normal perfusion in control untreated ischemic kidneys. This study also confirmed in vivo the efficacy of propionyl-L-carnitine of modulating ischemia-reperfusion injury in a syngeneic rat kidney transplant model to the extent that post-transplant delayed graft function was mostly prevented.
Several mechanisms by which propionyl-L-carnitine protected ischemic kidney improving functional recovery during reperfusion have been explored.
In the IPK preparation, perfusion pressure is maintained constant at 100 mm Hg by a flow pump; thus, the change in RPF is an indirect measurement of renal vascular resistance. During reperfusion RPF in kidneys pre-exposed to propionyl-L-carnitine was higher than in ischemic controls, indicating that renal vasodilation and enhancement of tissue perfusion contributed, at least in part, to the renoprotective effect of the substance. This is consistent with in vivo data in pigs that propionyl-L-carnitine increased post-ischemic blood flow of descending coronary artery22 and in vitro findings that the compound dilated human subcutaneous arteries through an endothelium-dependent mechanism23.
Propionyl-L-carnitine is one of the acyl derivatives of carnitine, a naturally occurring substance required for the transport of long-chain acyl groups derived from fatty acids across mitochondrial membranes, a necessary preliminary event to fatty acid oxidation and hence ATP production24,53. Depletion of this cofactor has been reported in several pathologic conditions, including myocardial ischemia54,55. Thus, the possibility exists that propionyl-L-carnitine may protect ischemic tissues by prolonging this oxidative metabolism and reducing the ischemia-induced loss of ATP. However, here we found that propionyl-L-carnitine had no effect on ATP levels in the ischemic kidneys. This is in line with previous reports in which both ATP levels and energy charge of ischemic hearts was not affected by propionyl-L-carnitine, despite significant protection toward ischemia-reperfusion injury in rat isolated perfused hearts37 and in open-chest anesthetized pigs22.
Others have proposed that propionyl-L-carnitine stabilizes plasma membrane during ischemia56. This effect may relate to propionyl-L-carnitine's ability to modify the molecular dynamics of the membrane bilayer region close to the glycerol backbone of phospholipids, as shown in an in vitro study on human red cell membranes21. Consistent with this interpretation are additional reports showing that propionyl-L-carnitine treatment of diabetic rats restored the ability of intact red cells to re-acylate membrane phosphatidylcholine57, and that this compound inhibited platelet-activating factor synthesis in human neutrophils and platelets20. That indeed propionyl-L-carnitine preserved cell membrane integrity is supported by our findings of consistent reduction of LDH release, a cell injury marker51,58, into the perfusate of IPKs exposed to ischemia/reperfusion. Moreover, the possibility that the protective action of propionyl-L-carnitine involved a cellular effect other than the energy-linked one after experimental ischemia rests also on the histological findings of very mild post-ischemic lesions both in the thick ascending limbs and in the proximal tubules in kidneys exposed to the drug, a pattern even better than that of non-ischemic control IPKs. This is supported also by in vivo data that propionyl-L-carnitine partially reduced tubular damage in syngeneic transplanted kidneys pre-exposed to four hours of cold ischemia.
The effects of propionyl-L-carnitine on membrane integrity and phospholipids also may account for attenuation of oxidant stress. Indeed, evidence is available that the compound protects from lipid peroxidation and free radical formation59,60. This suggests that infusion of carnitine derivatives into the renal artery may have prevented deterioration in renal function by limiting oxygen radical generation during ischemia-reperfusion. Indeed, immunohistochemical detection of 4-hydroxynonenal protein adducts, a major product of lipid peroxidation40,48,49, was very low in kidneys infused with propionyl-L-carnitine and exposed to ischemia/reperfusion as compared with untreated ischemic kidneys. Interestingly, reduction of 4-hydroxynonenal protein adduct generation mainly occurred at cortical and medullary tubules, the structures more susceptible to the ischemic-reperfusion injury. In line with these findings also are data that in IPKs, propionyl-L-carnitine completely prevented the increase of MDA concentration in the venous effluent after reperfusion.
By limiting free radical formation and preserving tubular cell integrity propionyl-L-carnitine also is expected to prevent the active inflammatory responses associated with parenchymal injury after ischemia-reperfusion through the release of cytokines and chemokines61. Available evidence shows that sublethally or even lethally injured proximal tubular and endothelial cells as a consequence of tissue hypoxia/reperfusion injury may act as a trigger of this inflammatory reaction61,62,63. In particular, polymorphonuclear cells (PMNs) recruited during reperfusion have long been implicated as critical mediators of the early renal parenchymal injury in ischemic acute renal failure64,65. PMNs, by amplifying an inflammatory response that leads to the generation of vasoconstrictor agents52, cytokines, and toxic mediators such as reactive oxygen species and proteases66, may ultimately induce additional damage in post-ischemic renal injury. Our results found that propionyl-L-carnitine added to the cold storage solution of kidneys undergoing four hours of cold ischemia before transplantation largely reduced early intragraft granulocyte infiltration. This finding indicates that, in addition to directly limiting free radical formation by renal parenchymal cells, propionyl-L-carnitine may have contributed to restore graft function by largely avoiding the additional tissue damage that would have been triggered by granulocytes recruited into the graft after ischemia-reperfusion.
It also should be taken into account that propionyl-L-carnitine has been shown to be beneficial by ameliorating ischemic damage to the mitochondria through inhibition of the toxic effect of amphiphilic compounds like lysophospholipids or long chain acylcarnitines9,19,67,68. This could be an additional mechanism by which propionyl-L-carnitine afforded protection in our models of renal ischemia-reperfusion injury.
In summary, propionyl-L-carnitine is of value in preventing the renal function deterioration that occurs after tissue ischemia-reperfusion injury in ex vivo isolated perfused rat kidney preparation as well as in a model of syngeneic kidney transplantation. The beneficial effect of propionyl-L-carnitine possibly relates to its ability of reducing lipid peroxidation and free radical generation, which contributes to the preservation of tubular cell structure and function as well as prevention of tissue inflammatory cell infiltration. Further studies, however, are required to get more insights on the relevant mechanisms through which propionyl-L-carnitine exerts its beneficial effect on renal ischemia-reperfusion injury. Nevertheless, these findings open perspectives for treatment to prevent post-transplant delayed graft function, which may improve patient outcome after renal transplantation.
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Acknowledgments
This work was partially supported by a grant from Sigma-Tau. We thank Dr. Nerina Corsico (Sigma-Tau, Industrie Farmaceutiche Riunite S.p.a., Rome, Italy) for kindly providing propionyl-L-carnitine and L-carnitine, and Mr. Alessandro Peschechera for performing the ATP measurements. The authors also thank Dr. Agnieszka Czauz for technical assistance, Mr. Gianfranco Marchetti for the work on tissue histology, and Ms. Daniela Rottoli for technical assistance. Ms. Federica Casiraghi helped to prepare the manuscript.
