Sandra van den Engel

Short‐term dietary restriction and fasting precondition against ischemia reperfusion injury in mice

Dietary restriction (DR) extends lifespan and increases resistance to multiple forms of stress, including ischemia reperfusion injury to the brain and heart in rodents. While maximal effects on lifespan require long‐term restriction, the kinetics of onset of benefits against acute stress is not known. Here, we show that 2–4 weeks of 30% DR improved survival and kidney function following renal ischemia reperfusion injury in mice. Brief periods of water‐only fasting were similarly effective at protecting against ischemic damage. Significant protection occurred within 1 day, persisted for several days beyond the fasting period and extended to another organ, the liver. Protection by both short‐term DR and fasting correlated with improved insulin sensitivity, increased expression of markers of antioxidant defense and reduced expression of markers of inflammation and insulin/insulin‐like growth factor‐1 signaling. Unbiased transcriptional profiling of kidneys from mice subject to short‐term DR or fasting revealed a significant enrichment of signature genes of long‐term DR. These data demonstrate that brief periods of reduced food intake, including short‐term daily restriction and fasting, can increase resistance to ischemia reperfusion injury in rodents and suggest a rapid onset of benefits of DR in mammals.

Angiotensin-(1–7) and the G Protein-Coupled Receptor Mas Are Key Players in Renal Inflammation

Angiotensin (Ang) II mediates pathophysiologial changes in the kidney. Ang-(1–7) by interacting with the G protein-coupled receptor Mas may also have important biological activities. In this study, renal deficiency for Mas diminished renal damage in models of renal insufficiency as unilateral ureteral obstruction and ischemia/reperfusion injury while the infusion of Ang-(1–7) to wild-type mice pronounced the pathological outcome by aggravating the inflammatory response. Mas deficiency inhibited NF-κB activation and thus the elevation of inflammation-stimulating cytokines, while Ang-(1–7) infusion had proinflammatory properties in experimental models of renal failure as well as under basal conditions. The Ang-(1–7)-mediated NF-κB activation was Mas dependent but did not involve Ang II receptors. Therefore, the blockade of the NF-κB-activating properties of the receptor Mas could be a new strategy in the therapy of failing kidney.

Differential Expression of Heme Oxygenase-1 and Vascular Endothelial Growth Factor in Cadaveric and Living Donor Kidneys after Ischemia-Reperfusion

The extent of graft damage after ischemia-reperfusion reflects the balance between deleterious events and protective factors. Heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) may contribute to cytoprotection by their anti-inflammatory and antiapoptotic properties. For investigating whether HO-1 and VEGF play a role in the adaptive response to ischemia-reperfusion injury after renal transplantation, kidney biopsies were analyzed from living (n = 45) and cadaveric (n = 16) donors, obtained at three time points: at the end of cold storage T(-1), after warm ischemia T(0), and after reperfusion T(+1). The mRNA expression levels of HO-1, VEGF(165), Bcl-2, Bax, and hypoxia inducible factor-1alpha were quantified by real-time reverse transcriptase-PCR, and the HO-1 and VEGF proteins were analyzed by immunohistochemistry. Cadaveric donor kidneys presented higher mRNA expression levels of hypoxia inducible factor-1alpha. In contrast, mRNA expression levels of HO-1, VEGF(165), and Bcl-2 were significantly lower in kidneys from cadaveric donors. Overall, a significant correlation was observed between mRNA expression of Bcl-2 and VEGF(165), between Bcl-2 and HO-1, and between HO-1 and VEGF(165). Moreover, protein expression of HO-1 and VEGF was detected in the same anatomical kidney compartments (glomerulus, arteries, and distal tubules). Renal function at the first week posttransplantation (analyzed by serum creatinine levels) showed a significant correlation with both HO-1 and VEGF mRNA expression, reinforcing the protective role of both genes in the early events of transplantation. It is concluded that the lower expression of HO-1, VEGF(165), and Bcl-2 in cadaveric donor kidneys can reflect a defective adaptation against ischemia-reperfusion injury that may affect their function in the short term.

Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

Cockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging‐like symptoms of this disorder include dysmyelination within the central nervous system, progressive sensineuronal hearing loss and retinal degeneration. We tested the effects of congenital nucleotide excision DNA repair deficiency on acute oxidative stress sensitivity in vivo. Surprisingly, we found mouse models of Cockayne syndrome less susceptible than wild type animals to surgically induced renal ischemia reperfusion injury, a multifactorial injury mediated in part by oxidative damage. Renal failure‐related mortality was significantly reduced in Csb−/– mice, kidney function was improved and proliferation was significantly higher in the regenerative phase following ischemic injury. Protection from ischemic damage correlated with improved baseline glucose tolerance and insulin sensitivity and a reduced inflammatory response following injury. Protection was further associated with genetic ablation of a different Cockayne syndrome‐associated gene, Csa. Our data provide the first functional in vivo evidence that congenital DNA repair deficiency can induce protection from acute stress in at least one organ. This suggests that while specific types of unrepaired endogenous DNA damage may lead to detrimental effects in certain tissues, they may at the same time elicit beneficial adaptive changes in others and thus contribute to the tissue specificity of disease symptoms.

A comparison of inflammatory, cytoprotective and injury gene expression profiles in kidneys from brain death and cardiac death donors.

Background The superior long-term survival of kidneys from living donors (LDs) compared with kidneys from donation-after-brain-death (DBD) and donation-after-cardiac-death (DCD) donors is now well established. However, comparative studies on transcriptional changes that occur at organ retrieval and during and after cold ischemia (CI) are sparse. Methods Using a rat model, we used qRT-PCR to examine expression levels of inflammatory, cytoprotective, and injury genes at different time points after organ retrieval. Cleaved caspase-3 was used to evaluate early apoptosis in DCD and DBD kidneys. Results Immediately after retrieval, we found massive up-regulation of proinflammatory genes interleukin-1&bgr;, interleukin-6, tumor necrosis factor-&agr;, monocyte chemotactic protein-1, P-selectin, and E-selectin in DBD compared with LD and DCD kidneys. A significant increase in the expression of injury markers Kim-1, p21, and the cytoprotective gene heme oxygenase-1 accompanied this. Bax was increased in DCD kidneys, and Bcl-2 was decreased in DBD kidneys. After 2 hr of CI in the LD group and 18 hr in the DBD and DCD groups, gene expression levels were similar to those found after retrieval. During 18 hr of cold storage, expression levels of these genes did not change. In DCD and DBD kidneys, early apoptosis increased after CI. Discussion/Conclusion The gene expression profile in DBD kidneys represents an inflammatory and injury response to brain death. In contrast, DCD kidneys show only mild up-regulation of inflammatory and injury genes. These results may imply why delayed graft function in DCD kidneys does not have the deleterious effect it has on DBD kidneys.

Amelioration of renal ischaemia-reperfusion injury by synthetic oligopeptides related to human chorionic gonadotropin

BACKGROUND We have previously reported that small synthetic oligopeptides related to human beta-chorionic gonadotropin (beta-hCG) can reduce inflammation. Here we investigated whether such oligopeptides can reduce renal ischaemia-reperfusion injury in the mouse. METHODS Ten different oligopeptides were administered 1 min before induction of renal ischaemia and 1 min before reperfusion. RESULTS Survival at 72 h post-reperfusion was significantly higher in mice treated with oligopeptides MTRV, LQG, VLPALPQ or AQGV as compared to placebo-treated mice. Some oligopeptides were more effective than others. AQGV completely prevented mortality and best preserved kidney function. Next, AQGV was tested in a dose-escalating study in a range of 0.3-30 mg/kg. A survival gain was observed with all doses. Improvement of kidney function was observed from 1 mg/kg. Highest survival and best preserved kidney function were observed at 3 and 10 mg/kg. Upon treatment with AQGV, a significantly lower influx of neutrophils was found, apoptosis was decreased, whereas tubular epithelial cell proliferation was significantly increased at 24 h post-reperfusion. Serum levels of TNF-alpha, INF-gamma, IL-6 and IL-10 were significantly decreased at 24 h post-reperfusion. E-selectin mRNA levels in kidneys were significantly decreased at 6 h post-reperfusion. AQGV did not reduce mortality when treatment was started after reperfusion. CONCLUSIONS This study shows that small oligopeptides related to the primary structure of beta-hCG, especially AQGV, are promising potential drugs for preventing the development of renal ischaemia-reperfusion injury.

Glucose supplementation does not interfere with fasting-induced protection against renal ischemia/reperfusion injury in mice.

Background. Preoperative fasting induces robust protection against renal ischemia/reperfusion (I/R) injury in mice but is considered overcautious and possibly detrimental for postoperative recovery in humans. Furthermore, fasting seems to conflict with reported benefits of preoperative nutritional enhancement with carbohydrate-rich drinks. Here, we investigated whether preoperative ingestion of a glucose solution interferes with fasting-induced protection against renal I/R injury. Methods. Mice were randomized into the following groups: fasted for 3 days with access to water (fasted) or a glucose solution (fasted+glc) and fed ad libitum with water (fed) or a glucose solution (fed+glc). After induction of bilateral renal I/R injury, all animals had free access to food and water. Calorie intake, body weight, insulin sensitivity, kidney function, and animal survival were determined. Results. Fed+glc mice had a comparable daily calorie intake as fed mice, but 50% of those calories were obtained from the glucose solution. Fasted+glc mice had a daily calorie intake of approximately 75% of the intake of both fed groups. This largely prevented the substantial body weight loss seen in fasted animals. Preoperative insulin sensitivity was significantly improved in fasted+glc mice versus fed mice. After I/R injury, kidney function and animal survival were superior in both fasted groups. Conclusions. The benefits of fasting and preoperative nutritional enhancement with carbohydrates are not mutually exclusive and may be a clinically feasible regimen to protect against renal I/R injury.

Inflammatory genes in rat livers from cardiac- and brain death donors

BACKGROUND Liver transplantation (LT) is the only life-saving treatment for patients with end-stage liver disease. The increase in patients has prompted the use of not only donation after brain death (DBD) donors but also living donors (LD) and donation after cardiac death (DCD) donors. Donor-type affects early graft function and graft survival as evidenced by an increased risk of developing ischemic type biliary lesions and higher risk of graft loss in DCD as compared with those in DBD grafts. METHODS Using a rat model, we used quantitative reverse transcription-polymerase chain reaction to examine expression levels of proinflammatory, cytoprotective, and injury genes and determined apoptosis in DCD and DBD livers at different time points after retrieval. RESULTS After retrieval, early mediators of inflammation MCP-1, HMGB1, and toll-like receptor (TLR 4) were increased in DCD livers, whereas the proinflammatory genes interleukin 6, interleukin 1β, tumor necrosis factor alpha, P-selectin, and E-selectin were massively upregulated in DBD compared with those in LD livers. HO-1 was increased in both postmortem groups. After cold ischemia, DCD livers showed increased levels of MCP-1, TLR4, and HMGB1, whereas expression of proinflammatory genes in DBD liver remained high. During 12 h of cold storage, expression levels remained stable except Hif-1α and HMGB1. DCD showed higher number of apoptotic cells compared with DBD livers. CONCLUSIONS Compared with LD, DCD livers showed only mild upregulation of inflammatory markers, but increased levels of MCP-1, HMGB1, and TLR4, and more apoptotic cells. In contrast, DBD livers showed a massive inflammatory response. These differences in tissue injury and inflammatory response might be relevant for the outcome after LT.

Abstract 4417: Fasting protects against the adverse side effects of chemotherapy but has no effect on antitumor activity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The multi-modality approach to colorectal liver metastases has led to an increase in the number of patients eligible for hepatic resection. The tumor response to chemotherapeutic drugs plays a pivotal role in the success of this approach. The limitations to their use are often severe adverse side-effects, resulting in an early discontinuation of the chemotherapy. In previous work we have shown that fasting leads to increased expression of cytoprotective and antioxidant genes. In this study we have examined the effects of fasting prior to administration of a high dose of the chemotherapeutic agent irinotecan on the occurrence of chemotherapy-associated adverse events and anti-tumor effect in C26 coloncarcinoma bearing mice. Male BALB/c mice were subcutaneously implanted with a 15 mm3 cube of C26 coloncarcinoma cells and divided into 4 groups (n=6/group). Ten days after implantation, 2 groups were fasted for 72 hours and 2 groups were fed ad libitum. After 72 hours all groups were fed ad libitum again. One ad libitum group and one fasting group were treated with a cumulative dose of 400 mg/kg irinotecan ip, administered on days 0, 2 and 4 relative to fasting. Tumor growth was measured daily using Vernier calipers. Ocurrence of adverse side effects was recorded daily. Leucocytes were counted on day 8 after the first irinotecan dose. All mice were sacrificed 10 days after the first irinotecan injection or earlier when tumor volume exceeded allowable range. Tumors were resected, measured and weighted. In the ad libitum fed group mice showed weight loss from the first irinotecan injection, while the fasted mice gained weight during the observation period. In the ad libitum fed mice other adverse side effects were observed from day 4 after the first irinotecan gift. They displayed different behaviour, reduced mobility, had ruffled hair, a hunched posture and diarrhea. The fasted mice showed no visible adverse side effects. The number of leukocytes in the fasted group treated with irinotecan was significantly higher, than in the ad libitum fed treated animals (6.5*10ˆ6/mL vs. 3.2*10ˆ6/mL, p<0.001). The experiment had to be terminated before the end of the observation period in 67% of the non-treated mice due to progressive tumor growth. In both irinotecan treated groups tumor growth was similarly suppressed compared with the fasted and ad libitum fed groups without irinotecan (1271 mg vs. 2106 mg, p<0.001). Our data demonstrate that 72 hours of fasting prior to treatment with a high dose of irinotecan prevents the occurrence of adverse side effects, while the antitumor activity is not affected. These data suggest that a preoperative fasting regimen may improve the therapeutic index of chemotherapeutic agents and increases the efficacy of chemotherapeutic treatment. Citation Format: Sander A. Huisman, Sandra van den Engel, Henk P. Roest, Jan N.M. IJzermans, Ron W.F. de Bruin. Fasting protects against the adverse side effects of chemotherapy but has no effect on antitumor activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4417. doi:10.1158/1538-7445.AM2013-4417

Preoperative fasting protects aged-corpulent mice against renal ischemia-reperfusion injury

Background Oxidative stress (OS), the production of free oxygen radicals caused by for instance renal ischemia/reperfusion injury (IRI), results in age associated diseases and accelerated aging. We have shown that preoperative fasting in young-lean C57BL6 male mice protects against renal IRI [1]. Since human patients are usually older and suffer from (co)morbidities, we investigated the effects of preoperative fasting on OS induced by renal IRI in both female and male aged-corpulent mice in a F1 -FVB/C57BL6hybrid background.