Gamma-glutamyl transpeptidase. A sensitive indicator of renal ischaemic injury in experimental animals and renal homograft rejection in man.

The sites of ischaemic injury within the kidney are reviewed and the diagnostic value of measurements of plasma and urinary enzymes in renal ischaemic injury and in renal homotransplant rejection in experimental animals and man is examined. Gamma-glutamyl transpeptidase (gamma-GT) is an enzyme primarily located in the brush border of the proximal convoluted tubule of the kidney. Its unique localization in the cells most easily damaged by ischaemia and its ease of assay provide the rationale for its use in the measurement and diagnosis of renal ischaemic injury. gamma-GT activity was measured in dogs undergoing varying periods of renal ischaemia and under conditions of local renal hypothermia and was shown to be a sensitive indicator of ischaemic injury. Twenty consecutive patients undergoing renal homotransplantation were studied by daily estimation of their 24-h urinary gamma-GT activity; excellent correlation was obtained between raised levels of this enzyme and the clinical diagnosis of transplant rejection.     

Role of PI3-kinase/Akt signalling pathway in renal function and cell proliferation after renal ischaemia/reperfusion injury in mice

BACKGROUND: PI3K/Akt pathway has been shown to play a critical role in the regulation of mitogenic signalling, apoptosis, cell proliferation and survival in a variety of cells and tissues. The aim of the present study was to investigate the role of PI3K/Akt pathway in the renal ischaemia/reperfusion. METHODS: Four experimental groups, sham-operative mice, vehicle-delivered and wortmannin-treated ischaemic/reperfusion injury mice, wortmannin-treated normal mice were designed to examined serum blood urea nitrogen level, renal injury, proliferating cell nuclear antigen protein and Akt phosphorylation status at 30 min, 90 min, 24 h, 48 h of reperfusion after ischaemic treatment. Wortmannin or its vehicle was given intraperitoneally at 4 h before surgery. Blood urea nitrogen was measured, and immunohistochemistry and western blotting were used to detect the components of PI3K/Akt pathway in the ischaemic/reperfusion injury kidney. RESULTS: PI3-kinase inhibitor wortmannin imposes a deleterious effect on serum blood urea nitrogen level, renal function after renal ischaemia/reperfusion injury in mice. The renal cell proliferation increased after ischaemia/reperfusion injury in mouse, which could be inhibited by wortmannin. Phosphorylation of Akt was increased after ischaemia/reperfusion in the mouse kidney, and reduced by wortmannin administration. CONCLUSION: This primary study suggests that PI3-kinase/Akt signalling pathway play an important role in the regulation of the renal repair after ischaemia/reperfusion injury.     

Urinary gamma-glutamyl transpeptidase, an indicator of renal ischaemic injury and homograft rejection.

Gammaglutamyl transpeptidase is an enzyme primarily located in the brush border of the proximal convoluted tubules of the kidney. Its unique localisation in the renal cells most easily damaged by ischaemia and its ease of assay provides the rationale for its use in the measurement of renal ischaemic injury. Using a standard experimental animal model, canine urinary gamma-GT activity was shown to be increased up to 70-fold following 90 min of unilateral renal ischaemia and was significantly raised following only 5 min ischaemia. The urinary gamma-GT was used as a measure of ischaemic injury associated with renal transplantation in man and 20 consecutive patients undergoing kidney transplant were studied by daily 24-hour urinary gamma-GT estimations and excellent correlation was obtained between raised enzyme activity and the clinical diagnosis of transplant rejection.     

Localization of renal oxidative stress and inflammatory response after lithotripsy

OBJECTIVE

To determine if the acute renal oxidative stress and inflammation after extracorporeal shock wave lithotripsy (ESWL), thought to be mediated by ischaemia, is most severe in the portion of the kidney within the focal zone of the lithotripter, and if these effects result primarily from ischaemic injury.

MATERIALS AND METHODS

Pigs (7–8‐weeks old) received either 2000 shock waves at 24 kV to the lower‐pole calyx of one kidney or unilateral renal ischaemia for 1 h. A third group (sham) received no treatment. Timed urine and blood samples were taken for analysis of lipid peroxidation and the inflammatory cytokines, tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). At 4 h after treatment, kidneys were removed and samples of cortex and medulla were frozen for analysis of cytokines and heme oxygenase‐1 (HO‐1).

RESULTS

ESWL did not affect urinary excretion of malondialdehyde, but did elicit an eight‐fold induction of HO‐1 in the portion of the renal medulla within the focal zone of the lithotripter (F2), while remaining unchanged elsewhere in the treated kidney. There was no induction of HO‐1 in renal tissue after ischaemia‐reperfusion. Urinary excretion of TNF‐α increased from the lithotripsy‐treated kidney by 1 h after treatment, but was unaffected by ischaemia‐reperfusion. As with the HO‐1 response after lithotripsy, IL‐6 increased only in the renal medulla at F2. By contrast, ischaemia‐reperfusion increased IL‐6 in all samples from the treated kidney.

CONCLUSION

These findings show that the acute oxidative stress and inflammatory responses to ESWL are localized to the renal medulla at F2. Furthermore, the differing patterns of markers of injury for ESWL and ischaemia‐reperfusion suggest that ischaemia is not the principal cause of the injury response after ESWL.     

The impact of warm ischaemia on renal function after laparoscopic partial nephrectomy

Authors from Cleveland assessed the impact of warm ischaemia on renal function, using their large database of laparoscopic partial nephrectomies for tumour. While agreeing that renal hilar clamping is essential for precise excision of the tumour, and other elements of the operation, the authors indicate that warm ischaemia may potentially damage the kidney. However, they found that there were virtually no clinical sequelae from warm ischaemic of up to 30 min. They also found that advancing age and pre-existing renal damage increased the risk of postoperative renal damage. OBJECTIVE: To assess the effect of warm ischaemia on renal function after laparoscopic partial nephrectomy (LPN) for tumour, and to evaluate the influence of various risk factors on renal function. PATIENTS AND METHODS: Data were analysed from 179 patients undergoing LPN for renal tumour under warm ischaemic conditions, with clamping of the renal artery and vein. Renal function was primarily evaluated in two groups of patients: 15 with tumour in a solitary kidney, who were evaluated by serial serum creatinine measurements; and 12 with two functioning kidneys undergoing unilateral LPN, and evaluated by renal scintigraphy before and 1 month after LPN to quantify differential renal function. Also, in all 179 patients, mean serum creatinine data at baseline, 1 day after LPN, at hospital discharge, and at the last follow-up were provided as supportive evidence. Logistic regression analyses were used to assess the effect of various risk factors on renal function after LPN, i.e. patient age, baseline serum creatinine, tumour size, solitary kidney status, duration of warm ischaemia, pelvicalyceal suture repair, urine output and intravenous fluids during LPN. RESULTS: In the group of patients with a solitary kidney the mean warm ischaemia time was 29 min, kidney parenchyma excised 29%, and serum creatinine at baseline, discharge, the peak after LPN and at the last follow-up (mean 4.8 months) 1.3, 2.3, 2.8, and 1.8 mg/dL, respectively. One patient (6.6%) required temporary dialysis. In the second group, assessed by renal scintigraphy, the function of the operated kidney was reduced by a mean of 29%, commensurate with the amount of parenchyma excised. For all 179 patients, a combination of age > or = 70 years and a serum creatinine level after LPN of > or = 1.5 mg/dL correlated with a higher serum creatinine after LPN. On logistic regression, baseline serum creatinine and solitary kidney status were the only variables significant for serum creatinine status after LPN. CONCLUSIONS: The bloodless field provided by renal hilar clamping is important for precise tumour excision, pelvicalyceal suture repair and securing parenchymal haemostasis during LPN. However, renal hilar clamping causes warm ischaemia. These data indicate that the clinical sequelae of warm ischaemic renal injury of approximately 30 min are minimal. Advancing age and pre-existing azotaemia increase the risk of renal dysfunction after LPN, especially when the warm ischaemia exceeds 30 min.     

Low arginine plasma levels do not aggravate renal blood flow after experimental renal ischaemia/reperfusion.

BACKGROUND: Ischaemic renal dysfunction is present in many clinical settings, including cardiovascular surgery. Renal hypoperfusion seems to be the most important pathophysiologic mechanism. Arginine plasma levels are rate limiting for NO synthesis, and low arginine plasma levels are seen after major vascular surgery. OBJECTIVE: to establish the effects of low arginine plasma levels on renal blood flow after renal ischaemia/reperfusion. DESIGN: Wistar rats were used in this unilateral renal ischaemia/reperfusion model. After 70 min of ischaemia, the kidney was reperfused for 150 min. Arginase infusion was used to lower arginine plasma levels. Blood flow measurement was performed at the end of the experiment using radiolabelled microspheres. Additional experiments were performed for histopathology. RESULTS: Arginase efficiently decreased arginine plasma levels to about 50% of normal. There was a lower blood flow in the ischaemic kidney than the contralateral (non-ischaemic) kidney. Lowering arginine plasma levels did not reduce renal blood flow in the ischaemic kidney. Renal histopathology was not influenced by lowered arginine plasma levels. CONCLUSIONS: Lowering arginine plasma levels did not affect blood flow or histology following renal ischaemia and reperfusion.     

Macrophages contribute to the development of renal fibrosis following ischaemia/reperfusion-induced acute kidney injury.

BACKGROUND: Ischaemia/reperfusion is a major cause of acute kidney injury and can result in poor long-term graft function. Although most of the patients with acute kidney injury recover their renal function, significant portion of patients suffer from progressive deterioration of renal function. A persistent inflammatory response might be associated with long-term changes following acute ischaemia/reperfusion. Macrophages are known to infiltrate into tubulointersitium in animal models of chronic kidney disease. However, the role of macrophages in long-term changes after ischaemia/reperfusion remains unknown. We aimed to investigate the role of macrophages on the development of tubulointerstitial fibrosis and functional impairment following acute ischaemia/reperfusion injury by depleting macrophages with liposome clodronate. METHODS: Male Sprague-Dawley rats underwent right nephrectomy and clamping of left renal vascular pedicle or sham operation. Liposome clodronate or phosphate buffered saline was administered for 8 weeks. Biochemical and histological renal damage and gene expression of various cytokines were assessed at 4 and 8 weeks after ischaemia/reperfusion. RESULTS: Ischaemic/reperfusion injury resulted in persistent inflammation and tubulointerstital fibrosis with decreased creatinine clearance and increased urinary albumin excretion at 4 and 8 weeks. Macrophage depletion attenuated those changes. This beneficial effect was accompanied with a decrease in gene expression of inflammatory and profibrotic cytokines. CONCLUSIONS: These results suggest that macrophages play an important role in mediating persistent inflammation and fibrosis after ischaemia/reperfusion leading to a development of chronic kidney disease. Strategies targeting macrophage infiltration or activation can be useful in the prevention of development of chronic kidney disease following ischaemic injury.     

In situ activation pattern of Met docking site following renal injury and hypertrophy.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) binds to its tyrosine kinase receptor, Met, thereby stimulating diverse cellular responses. The multifunctional docking site in the C-terminal domain mediates the signal of phosphorylated Met receptors to multiple transducers. The tyrosine at position 1356 of the Met docking site is crucial for cell motility and morphogenesis. METHODS: We examined the in situ distribution patterns of the Tyr1356-phosphorylated form of Met with a novel monoclonal antibody following renal injury and renal hypertrophy in rats. Sections of the kidney following either sham operation, transient ischaemia of one kidney or unilateral nephrectomy were analysed using indirect immunofluorescence staining and confocal laser scanning microscopy analysis of total Met protein levels and Tyr1356-phosphorylated Met (Met and pMet, respectively). RESULTS: At 6 h post-treatment, pMet increases in ischaemic kidneys compared with sham-operated kidneys, and these changes become substantial after 48 h in both medulla and cortex of ischaemic kidneys (P < 0.001). We also show significant up-regulation of Met predominantly in the medulla of ischaemic kidneys, 48 h following injury (P < 0.009). Inter-estingly, the stimulus for hypertrophy in the remnant kidney after uninephrectomy and the contra-lateral kidney during ischaemia is not accom-panied by significant up-regulation of Met or pMet staining compared with sham operation at both time points. CONCLUSIONS: We demonstrate in this work, for the first time, in situ detection of tyrosine kinase growth factor receptor docking site activation during pathological processes in the kidney. Using this methodology, we show a significant increase in Met docking site activity in both renal medulla and cortex solely following stimulation by ischaemia and repair.     

Polycystin-2 expression is increased following experimental ischaemic renal injury.

BACKGROUND: Mutations in PKD2 account for 15% of patients with autosomal dominant polycystic kidney disease. Expression of the PKD2 protein, polycystin-2, is developmentally regulated, suggesting a major role for this protein during nephrogenesis. However, the regulation of polycystin-2 expression in the adult kidney has not been previously explored. METHODS: We have utilized an established model of renal ischaemic injury to study polycystin-2 expression in adult rat kidney for up to 120 h following ischaemia. RESULTS: Our results indicate that polycystin-2 expression is increased in the post-ischaemic kidney by up to 5-fold, with a peak in expression at 48 h reperfusion. This time course mirrored the increase in cell proliferation observed. In the non-ischaemic kidney, polycystin-2 expression was highest in distal nephron segments but faint proximal tubular staining was also observed. No expression was seen in glomeruli. In the ischaemic kidney, polycystin-2 expression was greatly increased but the increase in expression was not restricted to segments with the highest number of proliferating cells. Moreover, polycystin-2 was detectable mainly intracellularly following ischaemia. Consistent with this, polycystin-2 was completely sensitive to endoglycosidase H during renal recovery, suggesting that it remains largely retained within the endoplasmic reticulum under these conditions. CONCLUSIONS: Our results provide the first evidence that polycystin-2 is increased following renal ischaemia, but show that this increase is not restricted to actively proliferating cells. The increase in polycystin-2 may relate instead to the process of cellular repair or differentiation following injury.     

Mesna: a novel renoprotective antioxidant in ischaemic acute renal failure.

BACKGROUND: Reactive oxygen species (ROS) play a key role in renal ischaemia-reperfusion injury. After establishing the in vitro anti-oxidative potential of mesna, a sulfhydryl-containing compound, its effect on kidney function and morphology in a rat model of ischaemic acute renal failure (ARF) was examined. METHODS: Mesna (180 mg/kg) was administered at different time points relative to ischaemia and/or reperfusion onset. Kidney function was assessed by glomerular filtration rate (GFR) and fractional sodium excretion (FE(Na)) before a 45-min period of unilateral renal artery clamping and following 90 min of reperfusion. Mesna was administered by bolus, 30 min before the induction of ischaemia, 5 min before ischaemia, 5 min before reperfusion, and 5 min after the onset of reperfusion. RESULTS: Mesna improved function of the ischaemic kidney at each administration. When mesna was administered 5 min before the onset of reperfusion, GFR reached 90-100% of its pre ischaemic value and FE(Na) was improved by 75%. The beneficial effect of mesna was also demonstrated by light and electron microscopy. Kidneys treated with mesna 5 min before reperfusion resembled ischaemic non-reperfused kidneys and showed subtle morphological and ultrastructural changes compared with ischaemic-reperfused kidneys. Mesna had no haemodynamic effect on renal blood flow and did not induce any osmotic diuresis. CONCLUSIONS: We suggest that mesna acts as an antioxidant. Its antioxidant potential together with optimal protection achieved when administered 5 min before reperfusion, supports the conclusion that mesna scavenges ROS generated at the onset of reperfusion, thus diminishing reperfusion injury and organ damage.     

Prevention of cold ischaemia-reperfusion injury by an endothelin receptor antagonist in experimental renal transplantation.

BACKGROUND: Endothelin (ET) is known to play a role in the pathogenesis of warm ischaemic renal damage, however, little is known about its involvement in renal cold ischaemia. This study was designed to investigate the response of ET after kidney cold ischaemia, and to assess the potential protective effect of bosentan, a dual, non-selective ET(A)/ET(B) receptor antagonist, against cold ischaemia reperfusion injury in a rat model of syngeneic renal transplantation. METHODS: Kidneys from Lewis rats were transplanted, either immediately or after 5 h of cold preservation. After 48 h, contralateral nephrectomy was performed. Rats were organized into three groups: Tr-NoISC, no cold ischaemia; Tr-ISC, 5 h cold ischaemia; and Tr-BOS, 5 h cold ischaemia plus bosentan (100 mg/kg/day, from the day before transplantation until the seventh day post-transplantation). On day 7, plasma and tissue immunoreactive ET (irET), as well as ET mRNA tissue expression, were evaluated. Renal function was measured by means of serum creatinine on days 3, 4, 5 and 7, and by creatinine clearance on day 7. Conventional histology was performed. RESULTS: The ischaemic group had significantly higher plasma irET levels than the non-ischaemic group and significantly lower levels than the bosentan group. Tissue irET levels and ET mRNA expression were similar in the ischaemic and bosentan groups and were higher than in the non-ischaemic group. Throughout the follow-up, serum creatinine was significantly higher in the ischaemic group than in the bosentan group. Moreover, creatinine decreased rapidly in the bosentan group after nephrectomy, whereas it continued to increase for 48 h in the ischaemic group. Kidneys from the ischaemic group showed a higher degree of tubular-cell necrosis and epithelial-cell detachment than kidneys from the bosentan group. CONCLUSIONS: We conclude that cold ischaemia and preservation damage induces an increase in renal ET mRNA and irET expression in the reperfusion phase, contributing both to the deterioration of renal function and to tubular necrosis. Bosentan is effective in protecting kidneys from this cold ischaemia reperfusion damage. Non-selective ET(A)/ET(B) receptor antagonists might be potentially useful in clinical renal transplantation.     

Remote ischaemic pre‐conditioning for the prevention of acute kidney injury

Acute kidney injury (AKI) is a common complication associated with high morbidity and mortality in hospitalized patients. One potential mechanism underlying renal injury is ischaemia/reperfusion injury (IRI), which attributed the organ damage to the inflammatory and oxidative stress responses induced by a period of renal ischaemia and subsequent reperfusion. Therapeutic strategies that aim at minimizing the effect of IRI on the kidneys may prevent AKI and improve clinical outcomes significantly. In this review, we examine the technique of remote ischaemic preconditioning (rIPC), which has been shown by several trials to confer organ protection by applying transient, brief episodes of ischaemia at a distant site before a larger ischaemic insult. We provide an overview of the current clinical evidence regarding the renoprotective effect of rIPC in the key clinical settings of cardiac or vascular surgery, contrast‐induced AKI, pre‐existing chronic kidney disease (CKD) and renal transplantation, and discuss key areas for future research.     

A novel method of induced renal hypothermia.

When the kidney is removed from cold storage for implantation into the recipient it gradually rewarms (second warm ischaemic time) and a prolonged second warm ischaemic time is a risk factor for delayed graft function. A cooling jacket has been designed to prevent this rewarming during transplantation. This study evaluates the efficacy of this device. Surface and core temperatures of less than 15 degrees Centigrade were maintained for 120 minutes. Renal function was significantly better in cooled than in uncooled kidneys in a single kidney canine model. Induced renal hypothermia, using a device such as this, should be a routine manoeuvre in renal transplantation.     

Protective effect of UR-12670 on chronic nephropathy induced by warm ischaemia in ageing uninephrectomized rats.

BACKGROUND: In young animals, renal ischaemia/reperfusion injury and mass reduction are associated with chronic lesions that mimic those found in chronic rejection. We have shown that the phospholipid platelet-activating factor (PAF) participates in young animals in such chronic nephropathy. Here we examine the long-term effects of the orally active PAF antagonist, UR-12670 in ageing uninephrectomized rats exposed to prolonged warm ischaemia. METHODS: Fifteen- to eighteen-month-old uninephrectomized male Sprague-Dawley rats were allocated into three groups and followed for 16 weeks: UNx, rats without ischaemia; UNxISC, ischaemic kidney (60 min), and UNxISC+UR, ischaemic kidney and UR-12670 from day 0 to the 16th week. Serum creatinine and proteinuria were monitored every 4 weeks. At the end of the study, conventional histology was performed and monocyte-macrophages were identified with the specific monoclonal antibody ED-1. RESULTS: The UNxISC group had severe acute renal failure with a high mortality rate, which was associated with incomplete restoration of renal function. Renal insufficiency in this group was sustained throughout the follow-up. Both UNx and UNxISC groups developed progressive proteinuria from the 12th week. Though UNxISC+UR group showed similar acute renal failure and mortality rate to the ischaemic non-treated group, serum creatinine decreased to levels similar to UNx group, which were maintained until the end of the study. Treatment of ischaemic kidneys with UR-12670 produced a slight decrease in 24-h proteinuria and a reduction in glomerulosclerosis, the mean tubulointerstitial score and number of monocyte-macrophages to values similar to UNx group. CONCLUSIONS: The chronic administration of the PAF antagonist UR-12670 attenuates the long-term effects of ischaemia-reperfusion injury in uninephrectomized ageing rats. The beneficial effect of this agent suggests that PAF contributes to the progression to late renal damage in this model.     

Amelioration of post-ischaemic renal injury by contralateral uninephrectomy: a role of endothelin-1.

BACKGROUND: Previous studies showed that unilateral renal damage is attenuated by prior contralateral uninephrectomy (Nx) in ischaemia-induced acute renal failure (ARF). Since renal ischaemia increases endothelin-1 (ET-1) production in the kidney, we examined whether the alteration of renal ET-1 content may contribute to the nephrectomy-induced attenuation of renal injury. METHODS: Ischaemic renal injury was provoked by 60-min left renal artery occlusion (RAO). Removal of the right kidney was performed just before RAO in the Nx group. Forty-eight hours after release of the clamp, renal ET-1 content was measured in both non-nephrectomized and unilaterally nephrectomized rats. We also examined the effects of a selective ET(A) receptor (FR139317) and monoclonal ET antibody (AwETN40) on the RAO-induced changes in renal haemodynamics at 2 and 48 h after RAO respectively. RESULTS: The plasma concentration of ET-1 did not change in the two groups of ARF rats, but the cortical content of ET-1 increased to a lesser extent in Nx animals after ischaemia. Prior removal of the right kidney significantly facilitated the percentage recovery of left renal blood flow (RBF) during the first 2 h after release of the clamp. The percentage recovery of inulin clearance (Cin) by the kidney was also significantly better in Nx than sham-Nx rats at 48 h after RAO. Continuous administration of FR139317 (50 mg/kg/day) using osmotic minipumps for 3 days significantly attenuated exogenous ET-1-induced decrease in Cin and RBF. Infusion of FR139317 restored the decrease in RBF to control values during first 2 h in sham-Nx rats. However, FR139317 and AwETN40 did not ameliorate the RAO-induced decline of Cin in sham-Nx or Nx rats at 2 and 48 h after ischaemia respectively. CONCLUSIONS: Contralateral uninephrectomy prior to ischaemia-induced ARF attenuated the increase in cortical ET-1 content and subsequent renal response to ischaemic injury. This beneficial effect of unilateral nephrectomy, however, was not mediated through well-preserved RBF due to reduced intrarenal ET-1 action.     

Antibody-induced modulation of the leukocyte CD11b integrin prevents mild but not major renal ischaemic injury.

BACKGROUND: CD11/CD18 beta(2) integrins are involved in leukocyte adhesion to the activated endothelium, and therefore represent a possible therapeutic target in the prevention of ischaemic acute renal failure (ARF). METHODS: To assess the effect of an anti-CD11b monoclonal antibody (mAb) in ischaemic ARF, uninephrectomized Fischer rats were subjected to 45 or 60 min of warm renal ischaemia, then received 1 mg of anti-CD11b mAb 5 min before reperfusion. RESULTS: After 45 min of ischaemia, renal function tests at 24 and 48 h were less altered in mAb-treated than in control rats, but after 60 min of ischaemia the same level of renal insufficiency was observed in the two groups. In parallel, milder tubular necrosis and less leukocyte infiltration were observed in the treated group after 45 min of ischaemia, but no difference was seen after 60 min compared to the control group. The mAb was detected on blood neutrophils up to 48 h after infusion and a marked down-regulation of CD11b expression on neutrophil surfaces was documented by flow cytometry. CONCLUSION: These results indicate that anti-CD11b mAb administered prior to reperfusion decreases moderate ischaemic ARF but fails to prevent renal injury secondary to prolonged ischaemia in this model.     

Murine renal ischaemia-reperfusion injury

SUMMARY:   Ischaemia/reperfusion is a major cause of acute kidney injury in native and transplant kidneys and is associated with significant morbidity and mortality. Murine models of renal ischaemia/reperfusion injury have great potential to improve understanding of the underlying processes and are an important focus of ongoing research into therapeutic and preventative strategies. Like all experimental models, murine models of renal ischaemia/reperfusion are prone to significant variability and results may be influenced by a number of technical and design factors. In this article we review the factors that may influence experimental results and provide a guide to conducting reproducible experiments in murine renal ischaemia/reperfusion.     

Failure of verapamil to protect from ischaemic renal damage

To reassess the reported protective effects of verapamil in renal ischaemia, we perfused the left kidney of uninephrectomized female Wistar rats with verapamil (0.1 and 1.0 mg/kg) immediately prior to clamping the renal artery for 60 min. When compared to controls, both doses of verapamil failed to afford protection with respect to urine flow, plasma creatinine, creatinine clearance or histology 24 and 48 h after ischaemia, whereas the purine nucleotide inosine (200 mg/kg) was partially protective: mean plasma creatinine 48 h after ischaemia (+/- SEM) was 547 +/- 54 mumol/l in controls, 686 +/- 38 mumol/l with 0.1 mg/kg verapamil, 491 +/- 68 mumol/l with 1.0 mg/kg verapamil and 374 +/- 45 mumol/l with inosine (p less than 0.05 vs. controls). Using the same model, the effect of verapamil 1.0 mg/kg on renal blood flow, creatinine clearance, urine flow and arterial pressure was studied in the first 2 h after ischaemia. Although significant amounts of verapamil were present in the kidney during ischaemia as evidenced by decreases in systemic blood pressure and in renal vascular resistance after unclamping the renal artery, the early course of renal failure was not altered when compared to controls. In conclusion, we have been unable to confirm earlier reports of a protective effect of verapamil in this rat model of ischaemic renal failure. If there is such an effect, its demonstration appears to depend on very specific experimental circumstances. Based on the results of this and other studies, verapamil is unlikely to afford an impressive overall benefit in the clinical setting of ischaemic renal failure.     

Pretreatment with granulocyte colony-stimulating factor attenuated renal ischaemia and reperfusion injury via activation of PI3/Akt signal pathway

Aim: Granulocyte colony-stimulating factor (G-CSF) has been shown to exert protective effects in various tissues and experimental models of ischaemia-induced injury. However, the mechanism of renoprotective action in ischaemia/reperfusion (I/R) renal injury of G-CSF was unknown. Methods: Male C57BL/6J mice, subjected to renal ischaemia for 45 min, 48 h and 7 days reperfusion, were administered either saline, wortmannin, G-CSF, and G-CSF plus wortmannin 3 days prior to I/R. Saline-treated group served as the control. At 48 h and 7 days of reperfusion, the mice were killed. Results: Significantly, renal dysfunction and morphological injury were identified at 48 h and 7 days after I/R. Wortmannin pretreatment worsened the renal injury significantly. However, G-CSF pretreatment significantly attenuated renal injury, reduced the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive ratio of renal tubular epithelial cells and inflammation cytokine expression in the kidney. Moreover, G-CSF pretreatment inhibited the expression of Bax and increased the expression of bcl-2 and p-Akt in the kidney. Wortmannin blunted the beneficial effects of G-CSF. Conclusion: The cytoprotective action of G-CSF against I/R injury seems to be associated with its anti-apoptotic action mediated by upregulation of p-Akt signal pathway.     

Ischaemic preconditioning protects the rat kidney from reperfusion injury.

OBJECTIVE: To examine the possible role of ischaemic preconditioning (IPC), an adaptive pathophysiological phenomenon that increases tolerance to ischaemia-reperfusion (I-R) injury, in renal protection when rats are presented with an I-R challenge. MATERIALS AND METHODS: Female Wistar rats (n=36) were divided randomly into four groups: (A) sham-operated controls; (B) IPC only; (C) renal ischaemia (RI) only; and (D) IPC+RI. The left kidney in groups B and D was preconditioned with four cycles of renal artery occlusion lasting 4 min, each occlusion separated by 11 min of reperfusion. The ischaemic insult, applied in groups C and D, comprised 40 min of sustained left renal artery occlusion. In Group D, the IPC cycle was completed 5 min before the start of the ischaemic insult. Differential left renal function was calculated by 99mTc-labelled dimercaptosuccinic acid scintigraphy at 0, 2 and 9 days after treatment, and expressed as a percentage of the total renal uptake. RESULTS: The mean (sem) maximum decrease in left renal function, to 14.5 (4.3)% of the total, occurred on day 2 in Group C. The equivalent value in Group D showed relative preservation of function, at 36.0 (3.5)% (P=0.001 compared with Group C). The mean left renal function improved by day 9, to 39.6 (6.7)% (Group C) and 48.6 (1.5)% (Group D). The mean left renal function in Group B (50.5-53.9%) did not differ from that in controls (49.4-51.4%). CONCLUSION: An IPC regimen applied 5 min before RI in the rat significantly protects it from the functional impairment associated with ischaemia and reperfusion.