EFFECT OF PENTOXIFYLLINE ON ISCHEMIC ACUTE RENAL FAILURE IN RABBITS

Abstract

Previous studies have demonstrated that levels of tumor necrosis factor-α (TNF-α) or its mRNA expression are increased in acute renal failure of various types including ischemia/reperfusion injury. This study was undertaken to determine whether pentoxifylline (PTX), an inhibitor of TNF-α production, provides a protective effect against ischemic acute renal failure in rabbits. Renal ischemia was induced by clamping bilateral renal arteries for 60 min. Animals were pretreated with PTX (30 mg/kg, i.v.) 10 min before release of clamp. At 24 h of reperfusion of blood after ischemia, changes in renal function, renal blood flow, and the expression of TNF-α mRNA were evaluated. Ischemia/reperfusion caused a marked reduction in GFR, which was accompanied by an increase of serum creatinine levels. Such changes were significantly attenuated by PTX pretreatment. PTX ameliorated the impairment of renal tubular function, but it had no effect on the reduction of renal blood flow induced by ischemia/reperfusion. The protective effect of PTX on functional changes was supported by morphological studies. The impairment of glucose and phosphate reabsorption in postischemic kidneys was associated with a depression in the expression of Na⁺-glucose and Na⁺-Pi transporters. The expression of TNF-α mRNA was increased after reperfusion, which was inhibited by PTX pretreatment. The PTX pretreatment in vitro prevented the release of lactate dehydrogenase induced by an oxidant t-butylhydroperoxide in rabbit renal cortical slices, but it did not produce any effect on the oxidant-induced lipid peroxidation, suggesting that PTX protection is not resulted from its antioxidant action. These results suggest that PTX may exert a protective effect against ischemic acute renal failure by inhibiting the production of TNF-α in rabbits.

• Ischemic-reperfusion injury
• Tumor necrosis factor-α
• Na⁺-cotransporter expression
• Rabbit kidney

INTRODUCTION

Tumor necrosis factor-α (TNF-α) is a cytokine with pleiotropic actions that participates in inflammation and immunity. Previous studies have demonstrated that levels of TNF-α or its mRNA expression are increased in acute nephrosis induced by aminonucleoside [[1]] and adriamycin [[2]], immune glomerulonephritis [3-4], diabetic nephropathy [[5]], and endotoxin-induced acute renal failure [[6]]. These reports suggest that TNF-α is linked to the pathogenesis of diverse renal diseases. TNF-α could be originated from both infiltrating monocytes and macrophages and resident glomerular cells. In vitro studies have shown that cultured rat and mouse mesangial cells produce TNF-α when exposed to adriamycin and puromycin [[2]]. Therefore, recent interest has focused on the participation of TNF-α in the mechanisms responsible for initiation and progression of glomerular injury [[6]].

Despite numerous experimental and clinical studies on the ischemic acute renal failure, the mechanisms underlying renal dysfunction following renal ischemia have not been clearly understood. One of the main functional changes in patients and experimental animals with ischemic acute renal failure is a decrease in glomerular filtration rate (GFR). The pathophysiologic mechanisms for the initiation and maintenance of the filtration failure have not been clearly elucidated [[7]]. Cytokines such as TNF-α may play a critical role in ischemic acute renal failure, since TNF-α has been implicated in the pathogenesis of glomerular injury [[6]].

Pentoxifylline (PTX) is a methylxanthine derivative with multiple hemorrheologic properties that has favorable effects on microcirculatory blood flow, but the precise mechanisms of its pharmacological action are not understood. Clinically, PTX has been used to treat intermittent claudication [[8]]. In addition, PTX has been known to decrease serum TNF-α levels [[5]], [[9]] and TNF-α mRNA expression [[10]]. This study was therefore undertaken to examine the effect of PTX on alterations in renal function in rabbits subjected to ischemia/reperfusion in order to evaluate the role of TNF-α in ischemic acute renal failure.

MATERIALS AND METHODS

Experimental Protocol

New Zealand White male rabbits weighing 1.5–2.5 kg were used. Anesthesia was induced by intramuscular injection of a mixture of ketamine (15 mg/kg) and xylazine (2.5 mg/kg) and maintained by sustaining doses (ketamine 5 mg/kg and xylazine 1 mg/kg) at intervals of approximately 30 min. A ventral midline incision was made and both renal arteries were clamped with nontraumatic vascular clamps for 60 min. The incision was closed and the animal was allowed to recover. Hartman solution was infused at a rate of 0.5 mL/min into an ear vein throughout the entire experimental period. After completion of surgery and recovery from anesthesia, the rabbits were returned to their metabolic cages. Urine was collected for 24 h prior to the induction of ischemia (the basal period) and for 24 h after ischemia (the postischemic period). On the same day, blood sample was collected from the ear artery. After 24 h of reflow, animals were sacrificed and the isolated kidneys were used for measurement of p-aminohippurate (PAH) uptake, Na⁺-K⁺-ATPase activity, and reduced glutathione (GSH). In experiments for PTX effect, the animals received PTX (30 mg/kg, i.v.) 10 min before the reflow of blood. The control animals untreated with the drug received an equal volume of saline.

Analysis and Calculation

Urine and blood samples were analyzed for Na⁺ (flame photometer, Beckman), creatinine, blood urea nitrogen (BUN), glucose, and phosphate (Sigma Chemical, St. Louis, MO). Glomerular filtration rate (GFR) was estimated from the endogenous creatinine clearance. Fractional Na⁺ excretion was calculated in the standard fashion. Alterations of renal function in postischemic kidney were estimated by comparing with the basal period of the same animal.

Renal blood flow was measured from a renal artery with a flowmeter (Transonic System Inc., NY) immediately before ischemia (basal) and 24 h after reflow.

Na⁺-K⁺-ATPase Activity Measurement

The renal cortical microsomal Na⁺-K⁺-ATPase activity was measured as described previously [[11]]. The microsomal fraction was prepared from cortex of postischemic kidneys. The ATPase activity of the microsomal fraction was determined by measuring inorganic phosphate (P_i) released by ATP hydrolysis during incubation of microsome with an appropriate medium containing 3 mM ATP (Sigma) as the substrate. The total ATPase activity was determined in the presence of 100 mM Na⁺, 20 mM K⁺, 3 mM Mg²⁺, 2 mM EDTA, and 40 mM imidazole (pH 7.4). The Mg²⁺-ATPase activity was determined in the absence of K⁺ and in the presence of 1 mM ouabain. The difference between the total and the Mg²⁺-ATPase activity was taken as a measure of the Na⁺-K⁺-ATPase activity. After a 5-min preincubation at 37°C, the reaction was initiated by the addition of the microsomal fraction. At the end of a 10-min incubation, the reaction was terminated by the addition of ice-cold 6% perchloric acid. The mixture was then centrifuged at 3,500 g, and P_i in the supernatant fraction was determined by the method of Fiske and SubbaRow [[12]].

Preparation of Renal Cortical Slices

New Zealand white male rabbits weighing 1.5 to 2 kg were sacrificed and the kidneys were removed rapidly. The kidneys were perfused immediately through the renal artery with an ice-cold isotonic saline solution containing 140 mM NaCl, 10 mM KCl and 1.5 mM CaCl₂ to remove as much blood as possible. Thin slices (0.25–0.35 mm) of renal cortex were prepared using a Stadie-Riggs microtome and were stored in an ice-cold modified Cross-Taggart medium containing 130 mM NaCl, 10 mM KCl, 1.5 mM CaCl₂, 5 mM glucose and 20 mM Tris/HCl (pH 7.4).

Uptake of PAH by Renal Cortical Slices

PAH uptake by renal cortical slices was measured in control and ischemic kidneys. After 24 h of reperfusion, both kidneys were removed and the renal artery was immediately perfused with an ice-cold isotonic saline solution containing 140 mM NaCl, 10 mM KCl and 1.5 mM CaCl₂, to remove as much blood as possible. Renal cortical slices were prepared as described above and were stored in an ice-cold modified Cross-Taggart medium containing 130 mM NaCl, 10 mM KCl, 1.5 mM CaCl₂, 5 mM Na acetate and 20 mM Tris/HCl (pH 7.8). Approximately 50 mg (wet wt.) of slices were then transferred into a 20 mL beaker containing 4 mL of the modified Cross-Taggart medium, and incubated with 75 μM ¹⁴C-PAH (Amersham, Alrington heights, IL). Incubation was carried out for 60 min in a Dubnoff metabolic shaker at 25°C under a 100% oxygen atmosphere. Immediately after incubation, the slices were quickly removed from the beaker, blotted, weighed and solubilized in 1 N NaOH. Aliquots of the incubation medium and the solubilized tissue were pipetted into a scintillation vial containing scintillation cocktail and the radioactivity was determined using a liquid scintillation counter (Packard Tricarb 300C). PAH uptake by renal slices was expressed as the slice to medium (S/M) ratio: the concentration of the compound in the tissue (mole/g wet tissue) divided by that in the medium (mole/mL medium).

Morphological Studies

Morphological evaluation was performed in kidneys subjected to 24 h reperfusion of blood following 60 min of ischemia. Kidneys were removed after intravascular perfusion with freshly prepared 1/2 Karnovsky solution. Following perfusion fixation, the samples were immersed in fixative for 4 h and processed for embedding in paraffin. Paraffin sections were stained with hematoxylin-eosin, examined, and photographed.

RNA Isolation and RT-PCR

Total cellular RNA was extracted from renal cortex using RNAzol B (Tel. Test. Inc., Friendswood, TX). RNA was quantified by spectrophotometry and formaldehyde gel electrophoresis, and stored at −80°C. Total RNA was reverse transcribed into first stand cDNA using oligo dT primer, and amplified by 35 cycles (94°C, 1 min; 52°C, 1 min; 72°C, 1 min) of polymerase chain reaction (PCR) using 20 pmole of specific primers. On completion of the PCR reaction, products were examined on 2% agarose gel. β-actin primers were used as an internal standard. The primers used for PCR are as follows:

Table

Name of DNA	Sequences	Size (bp)
β-Actin	5′-TTG TAA CCA ACT GGG ACG ATA TGG-3′ 5′-GAT CTT GAT CTT CAT GGT GCT AGG-3′	757
Na-Phosphate cotransporter	5′-TGG AGG AGG AGC AGA AGC CA-3′ 5′-GGA TGT TGA AGG AGG CGA CC-3′	533
Na-Glucose cotransporter	5′-TTCTTG GCG GGT CGG AGT ATG G-3′ 5′-TGG CTG GGA ATG GCT CGC AT-3′	553
TNF-α	5′-TAC TGA ACT TCG GGG TGA TTG GTC C-3′ 5′-CAG CCT TGT CCC TTG AAG AGA ACC-3′	305

Oxidant-Induced Injury in Renal Cortical Slices

Slices were incubated in a Cross-Taggart medium with 1.0 mM t-butylhydroperoxide (tBHP) for 60 min under a 100% O₂ atmosphere. The cell injury was estimated by measuring the release of lactate dehydrogenase (LDH). After incubation, tissues were homogenized in 2 mL of distilled water and the homogenate was centrifuged at 2,000 g for 5 min. The pellet was discarded and the supernatant was saved. LDH activity was determined in the supernatant and incubation medium using a LDH assay kit (Iatron Lab., Japan).

Lipid peroxidation was estimated by measuring the renal cortical content of malondialdehyde (MDA) according to the method of Uchiyama and Mihara [[13]]. Slices were homogenized in ice-cold 1.15% KCl (5% wt/vol). A 0.5 mL aliquot of homogenate was mixed with 3 mL of 1% phosphoric acid and 1 mL of 0.6% thiobarbituric acid. The mixture was heated for 45 min on a boiling water bath. After addition of 4 mL of n-butanol the contents were vigorously vortexed and centrifuged at 2,000 g for 20 min. The absorbency of the upper, organic layer was measured at 535 and 520 nm with a diode array spectrophotometer (Hewlett Packard, 8452A), and compared with freshly prepared malondialdehyde tetraethylacetal standards. MDA values were expressed as pmoles per mg protein. Protein was measured by the method of Bradford [[14]].

Statistical Analysis

The data are expressed as mean ± SEM and the difference between two groups was evaluated using Student's t-test. A probability level of 0.05 was used to establish significance.

RESULTS

Effect of PTX Pretreatment on Renal Function in Rabbits with Ischemic Acute Renal Failure

Table 1 summarizes changes in renal function in rabbits with ischemic acute renal failure treated and untreated with PTX. The serum creatinine level increased significantly after ischemia (3.20 ± 0.34 vs. 0.82 ± 0.12 mg/dL in the basal period). Similarly, the BUN level after renal ischemia was more than 4-fold greater than that observed in the basal period (146.84 ± 15.04 vs. 46.68 ± 7.38 mg/dL in the basal period). The PTX pretreatment significantly reduced the levels of serum creatinine and BUN after renal ischemia. PTX also attenuated the reduction of GFR in postischemic kidney. The average postischemic level of GFR in PTX-untreated animals was 0.09 ± 0.30 L/d/kg, which was less than 4% of the basal (2.34 ± 0.18 L/d/kg). The corresponding value in PTX-pretreated animals was 0.33 ± 0.06 L/d/kg, approximately 13.4% of the basal value (2.46 ± 0.03 L/d/kg). Renal ischemia induced an increase in fractional Na⁺ excretion (FE_Na) 5.5-fold in PTX-unpretreated animals (5.69 ± 0.18 vs. 1.03 ± 0.15% in the basal period), but it increased the FE_Na only 3.1-fold in PTX-pretreated animals (3.12 ± 0.02 vs. 1.00 ± 0.05% in the basal period).

Table 1. Effect of Pentoxifylline (PTX) on Renal Function in Rabbits with Ischemic Acute Renal Failure

Parameters	Periods	− PTX	+ PTX
Pcr (mg/dL)	Basal	0.82 ± 0.12	0.76 ± 0.15
	Postischemic	3.20 ± 0.34^*	1.99 ± 0.211-2^*#
BUN (mg/dL)	Basal	46.68 ± 7.38	46.36 ± 6.26
	Postischemic	146.84 ± 15.04^*	92.06 ± 14.821-2^*#
GFR (L/day/kg)	Basal	2.34 ± 0.18	2.46 ± 0.03
	Postischemic	0.09 ± 0.30^*	0.33 ± 0.061-2^*#
FENa (%)	Basal	1.03 ± 0.15	1.00 ± 0.05
	Postischemic	5.69 ± 0.18^*	3.12 ± 0.021-2^*#

Renal function was measured for 24 h before ischemia (basal period) and for 24 h of reperfusion following 60 min of ischemia (postischemic period) in rabbits treated and untreated with PTX. The animals received PTX (+PTX, 30 mg/kg, i.v.) or an equal volume of saline (− PTX) 10 min before reperfusion of blood. Data are mean ± SEM of nine animals in each group.

^*p < 0.05 compared with basal period;

^#p < 0.05 compared with the respective − PTX group.

Renal ischemia induced a marked increase in fractional excretion of glucose and inorganic phosphate, and the effect was significantly attenuated in PTX-pretreated animals (Fig. 1). PAH uptake by renal cortical slices and Na⁺-K⁺-ATPase activity in microsomal fraction prepared from cortical tissues were dramatically reduced by renal ischemia, and the reduction was significantly less in tissue preparation of PTX-pretreated animals (Figs. 2 and 3). The depletion of GSH content in renal tissue by ischemia was remarkedly prevented by PTX pretreatment (Fig. 4).

Figure 1. Effect of pentoxifylline (PTX) on fractional excretion of glucose (FE_Glucose, A) and inorganic phosphate (FE_Pi, B) in rabbits with ischemic acute renal failure. Data are mean ± SEM of nine animals in each group. ^*p < 0.05 compared with the respective basal value; ^#p < 0.05 compared with ischemia alone.

Figure 2. Effect of pentoxifylline (PTX) on p-aminohippurate (PAH) uptake by renal cortical slices in rabbits with ischemic acute renal failure. Data are mean ± SEM of nine animals in each group. ^*p < 0.05 compared with control; ^#p < 0.05 compared with ischemia alone.

Figure 3. Effect of pentoxifylline (PTX) on Na⁺-K⁺-ATPase activity in microsomal fraction from kidney cortex of rabbits with ischemic acute renal failure. Data are mean ± SEM of nine animals in each group. ^*p < 0.05 compared with control; ^#p < 0.05 compared with ischemia alone.

Figure 4. Effect of pentoxifylline (PTX) on glutathione (GSH) content in kidney cortex of rabbits with ischemic acute renal failure. Data are mean ± SEM of nine animals in each group. ^*p < 0.05 compared with control; ^#p < 0.05 compared with ischemia alone.

Effect of PTX on Renal Blood Flow

Figure 5 shows changes of renal blood flow in postischemic kidneys. The renal blood flow 24 h after reperfusion was markedly reduced, and the value was identical in PTX-pretreated and unpretreated animals.

Figure 5. Effect of pentoxifylline (PTX) on renal blood flow in rabbits with ischemic acute renal failure. Data are mean ± SEM of nine animals in each group. ^*p < 0.05 compared with the respective basal value.

Effect of PTX on Morphological Changes

Morphological evaluation was performed in postischemic kidneys. Figure 6A shows proximal tubules of control kidneys with normal microvilli appearance in brush border (arrows). When kidneys were subjected to ischemia, most of brush borders in the proximal tubule (T) were destructed (Fig. 6B). However, most of the brush borders of proximal tubules (T) in PTX-pretreated animals were regularly arranged (arrows) as in control kidneys (Fig.z 6C).

Figure 6. Morphological studies of pentoxifylline (PTX) effect on ischemic acute renal failure in rabbits. Samples were stained with hematoxylin-eosin. In control kidney (A), the proximal tubule shows normal structural appearance with plentiful brush border villi (arrows). In kidneys subjected to ischemia/reperfusion injury (B), brush borders of proximal tubule (T) were destructed. However, when kidneys were subjected to ischemia/reperfusion after PTX pretreatment (C), brush borders (arrows) of proximal tubule were regularly arranged as in control. Magnitude × 132.

Effect of PTX on Na⁺-glucose, Na⁺-Pi, and TNF-α mRNA

In previous study, we observed that ischemia causes marked reduction in reabsorption of glucose and phosphate in rabbits [[15]]. However, it was not determined whether such changes are associated with alterations in the expression of transporters. As shown in Figure 7 (upper panel), the expression of mRNA for Na⁺-glucose and Na⁺-Pi transporters was suppressed after ischemia. However, the expression of mRNA was increased in PTX-pretreated animals.

Figure 7. Effect of pentoxifylline (PTX) on expression levels of Na⁺-glucose (SG), Na⁺-phosphate (SP), TNF-α, and β-actin. Total RNAs were isolated from the cortex of kidneys subjected to ischemia/reperfusion with or without PTX pretreatment. Lane 1, 4, 7: control kidneys; Lane 2, 5, 8: ischemia; Lane 3, 6, 9; ischemia + PTX.

In order to evaluate the effect of PTX on TNF-α expression in kidneys with ischemic acute renal failure, the expression of TNF-α mRNA was measured in kidneys subjected to ischemia with or without PTX pretreatment. The expression of TNF-α mRNA was increased by ischemia/reperfusion and this was prevented by PTX (Fig. 7, lower panel).

Effect of PTX In Vitro on Oxidant-Induced Lipid Peroxidation in Renal Cortical Slices

Since PTX has been reported to inhibit the generation of reactive oxygen species [[16]] and scavenge hydroxyl radicals [[17]], the protective effect of PTX against the ischemia/reperfusion injury may be attributed to an inhibition of lipid peroxidation. To test this possibility, we examined the effect of PTX on the lipid peroxidation induced by an oxidant tBHP. As shown in Table 2, the tBHP-induced lipid peroxidation was not altered by PTX, but it was completely inhibited by 5 μM N,N′-diphenyl-p-phenylenediamine (DPPD), an antioxidant.

Table 2. Effect of Pentoxifylline (PTX) on Lipid Peroxidation Induced by t-Butylhydroperoxide (tBHP) in Renal Cortical Slices

Treatment	MDA Production (pmole/mg protein)
Control	133.29 ± 9.74
tBHP (1 mM)	517.20 ± 45.68^*
+PTX (0.05 mM)	403.00 ± 19.27^*
+PTX (0.1 mM)	550.18 ± 20.14^*
+DPPD (5 μM)	92.45 ± 23.29^#

Slices were treated with 1 mM tBHP in the presence or absence of PTX and DPPD for 60 min at 37°C. Data are mean ± SEM of four experiments.

^*p < 0.05 compared with control;

^#p < 0.05 compared with tBHP alone.

DISCUSSION

The present study demonstrated that the administration of PTX at a single dose of 30 mg/kg intravenously 10 min before the removal of arterial clamp provides a protective effect against the impairment of renal function in postischemic kidneys. The reduction in GFR and the increases in serum creatinine level and fractional Na⁺ excretion associated with the ischemia/reperfusion were significantly attenuated by PTX pretreatment (Table 1). PTX also prevented the proximal tubular dysfunction induced by ischemia as indicated by a significant protection of the impairments in renal glucose and phosphate reabsorption in vivo, PAH uptake by renal cortical slices, and Na⁺-K⁺-ATPase activity in microsomal fraction (Figs. 1-3. In addition, the GSH depletion in postischemic kidneys was significantly prevented by PTX pretreatment (Fig. 4). Such protective effects of PTX against proximal tubular dysfunction were supported by morphological studies (Fig. 6).

Renal blood flow usually remains reduced for a period of time beyond the initiation phase of ischemic acute renal failure in animal models [[18]]. Persistent vasoconstriction and cellular swelling cause poor blood reperfusion in the deep cortex and outer medulla after removal of the renal artery clamp [[19]]. These reports suggest that persistent medullary ischemia exacerbates the injury caused by the original insult. Indeed, an improvement of blood flow during the reperfusion period by any of several maneuvers reduces the severity of ischemic acute renal failure and accelerates recovery following ischemia [19-21]. PTX has been reported to stimulate the production of vasodilatory prostaglandins [[22]] and to interact with adenosine receptors [[23]] which have been known to increase the renal vascular resistance [[24]]. Thus, the protective effect of PTX against the ischemic acute renal failure could be due to a restoration of renal blood flow. However, the present study showed that renal blood flow in the postischemic period was less than 41% of that in the basal period and was not affected by PTX pretreatment (Fig. 5). These results may be consistent with the notion that restoration of renal blood flow in a postischemic kidney does not necessarily restore the GFR [[25]].

TNF-α is a potent proinflammatory cytokine capable of upregulating its own expression as well as the expression of other genes pivotal to the inflammatory response [[26]]. Furthermore, TNF-α causes a significant cellular damage and dysfunction via direct cytotoxicity and by recruitment of neutrophils and monocytes [[27]]. Ischemia/reperfusion induces TNF-α production in various cell types including the kidney in vivo [27-28] and in vitro [[29]]. Similarly, we found that TNF-α mRNA levels are increased in kidneys of rabbits with ischemic acute renal failure (Fig. 7). These data suggest that TNF-α may be an important mediator of ischemia/reperfusion injury. Modulation of TNF-α synthesis and activity might have important therapeutic implications. PTX administration has consistently been found to inhibit the production of TNF-α in animals and humans [[9]], [[30]] as well as to suppress TNF-α mRNA levels in in vitro studies [[10]]. In the present study, PTX decreased the expression of TNF-α mRNA and prevented the reduction of Na⁺-glucose and Na⁺-phosphate transporter mRNA expression in kidneys of rabbits with ischemic acute renal failure. In view of these findings, we suggest that PTX provides a protective effect against the ischemic acute renal failure in rabbits by inhibiting the TNF-α production.

Since PTX has been shown to inhibit the generation of leukocyte-derived reactive oxygen species in humans subjected to exercise [[16]] and scavenge hydroxyl radicals in vitro [[17]], the beneficial effect of PTX could be associated with its antioxidant action. In a previous study, however, we did not obtain any evidence supporting the involvement of reactive oxygen species in rabbits with ischemic acute renal failure [[31]], similar to others [32-33]. To determine if protection by PTX was the result of its antioxidant action, we examined the effect of PTX on the LDH release and lipid peroxidation induced by an oxidant tBHP in renal cortical slices. PTX protected partially against the LDH release induced by tBHP (Fig. 8). However, the tBHP-induced lipid peroxidation was not altered by PTX (Table 2). Taken together, it is unlikely that the protective effect of PTX against ischemic acute renal failure is associated with its antioxidant action.

Another important finding in the present study is the changes in the expression of Na⁺-dependent transporters in the proximal tubule. Renal ischemia has been demonstrated to cause a significant impairment in glucose reabsorption in rats [34-35] and rabbits [[15]]. Active reabsorption of glucose in the proximal tubule is driven by Na⁺-dependent transport systems [[36]]. However, it was not determined if ischemia/reperfusion-induced impairment in glucose reabsorption was the result of alterations in the expression of Na⁺-dependent transporter system. In this study, the reabsorption of glucose and phosphate in ischemia/reperfused kidneys was significantly impaired (Fig. 1). Such changes were accompanied by a reduction in the expression of Na⁺-glucose and Na⁺-phosphate transporters as evidenced by a decreased expression of their mRNAs (Fig. 7, upper panel).

In conclusion, PTX attenuated the reduction of GFR and the impairment of tubular function in rabbits with ischemic acute renal failure. Renal blood flow was decreased after ischemia/reperfusion, which was not altered by PTX pretreatment. PTX did not affect lipid peroxidation in renal cortical slices. Expression of TNF-α mRNA was increased after ischemia/reperfusion, which was inhibited by PTX. The beneficial effect of PTX on ischemic acute renal failure may be due to a suppression of the TNF-α production.