Preoperative fasting protects against renal ischemia-reperfusion injury in aged and overweight mice

Abstract

Ischemia-reperfusion injury (IRI) is inevitable during kidney transplantation leading to oxidative stress and inflammation. We previously reported that preoperative fasting in young-lean male mice protects against IRI. Since patients are generally of older age with morbidities possibly leading to a different response to fasting, we investigated the effects of preoperative fasting on renal IRI in aged-overweight male and female mice. Male and female F1-FVB/C57BL6-hybrid mice, average age 73 weeks weighing 47.2 grams, were randomized to preoperative ad libitum feeding or 3 days fasting, followed by renal IRI. Body weight, kidney function and survival of the animals were monitored until day 28 postoperatively. Kidney histopathology was scored for all animals and gene expression profiles after fasting were analyzed in kidneys of young and aged male mice. Preoperative fasting significantly improved survival after renal IRI in both sexes compared with normal fed mice. Fasted groups had a better kidney function shown by lower serum urea levels after renal IRI. Histopathology showed less acute tubular necrosis and more regeneration in kidneys from fasted mice. A mRNA analysis indicated the involvement of metabolic processes including fatty acid oxidation and retinol metabolism, and the NRF2-mediated stress response. Similar to young-lean, healthy male mice, preoperative fasting protects against renal IRI in aged-overweight mice of both genders. These findings suggest a general protective response of fasting against renal IRI regardless of age, gender, body weight and genetic background. Therefore, fasting could be a non-invasive intervention inducing increased oxidative stress resistance in older and overweight patients as well.

Figure 1. Relative body weight and survival.

Relative body weight (A) and survival (B) of male and female mice undergoing 37 and 60 minutes of renal ischemia-reperfusion injury (IRI), respectively, preceded by 3 day fasting or ad libitum feeding. In the first week after surgery, both fasted groups gradually lost weight after which they slowly gained weight in the weeks thereafter. Both fasted groups show a significantly improved survival: p = 0.0171 for males, p = 0.0040 for females. M = male, F = female, Adlib = ad libitum fed.

Figure 2. Kidney function after renal ischemia-reperfusion injury.

Kidney function of male and female mice after undergoing 37 and 60(A) Serum urea levels are significantly lower in fasted males and females on day 2 after renal IRI. (B) Serum creatinine levels show no significant differences between fasted and ad libitum fed mice. M = male, F = female, Adlib = ad libitum fed. Δ = no standard deviation is shown as only two animals comprised this group.

Figure 3. Histopathological analysis of kidneys after renal ischemia-reperfusion injury.

Histopathological analysis of kidneys of male and female mice after IRI. Mice sacrificed at the end of the experiment showed significantly less acute tubular necrosis (A) and significantly more tubular epithelial regeneration (B). Divided by intervention, both male and female fasted mice also showed less necrosis (C) and more regeneration (D). Fasted male mice sacrificed at day 7 showed similar pathology scores as the fasted mice at day 28 (mice indicated by the red symbols).*  =  significance (p<0.05) compared to the group ‘Found dead’, **  =  compared to the group ‘Moribund’, ***  =  compared to ad libitum fed males, ****  =  compared to ad libitum fed females.

Figure 4. Histological images of kidneys with acute tubular necrosis and tubular epithelial regeneration.

Representative images of HE stained kidney sections with acute tubular necrosis or tubular epithelial regeneration. (A) Multifocal severe acute tubular necrosis with typical diluted tubules, flattened epithelial lining and granular casts inside the tubules (arrows) in an ad libitum fed male mouse 4 days after 37 minutes of IRI. (B) On day 7, male fasted mice already show a high degree of regeneration (arrows) with minimal necrosis (stars). (C) Multifocal tubular regeneration as shown by mitosis bodies along the epithelial line (arrows) in a fasted male mouse 28 days after 37 minutes of IRI. (D) Multifocal severe acute tubular necrosis in ad libitum fed female mouse 4 days after 60 minutes of IRI. (E) Multifocal tubular regeneration in a fasted female mouse 28 days after 60 minutes of IRI.

Figure 5. Venn diagram and scatterplot of microarray data after fasting in young and aged mice.

A) Venn diagram of the significantly differentially up- and down-regulated (FDR ≤5%, FC ≥1.5) and overlapping probe sets in kidneys of three days fasted aged and young mice in comparison with normal fed control mice. B) Scatter plot comparing up- and down-regulated trends and their fold ratios of probe sets significantly regulated (FDR ≤5%) in young-lean (red), aged-overweight mice (black) or overlapping in both groups (green). Without fold change cut-off, 85.0% of the genes showed the same directionality in both age groups. The gray solid line represents the reference diagonal (ratio young  =  ratio aged); the gray dotted lines show the 1.5 fold change cutoffs applied in 5A.