Protein and Calorie Restriction Contribute Additively to Protection from Renal Ischemia Reperfusion Injury Partly via Leptin Reduction in Male Mice

Abstract

Background: Short-term dietary restriction (DR) without malnutrition preconditions against surgical stress in rodents; however, the nutritional basis and underlying nutrient/energy-sensing pathways remain poorly understood.

Objectives: We investigated the relative contribution of protein restriction (PR) vs. calorie restriction (CR) to protection from renal ischemia reperfusion injury (IRI) and changes in organ-autonomous nutrient/energy-sensing pathways and hormones underlying beneficial effects.

Methods: Mice were preconditioned on experimental diets lacking total calories (0-50% CR) or protein/essential amino acids (EAAs) vs. complete diets consumed ad libitum (AL) for 1 wk before IRI. Renal outcome was assessed by serum markers and histology and integrated over a 2-dimensional protein/energy landscape by geometric framework analysis. Changes in renal nutrient/energy-sensing signal transduction and systemic hormones leptin and adiponectin were also measured. The genetic requirement for amino acid sensing via general control non-derepressible 2 (GCN2) was tested with knockout vs. control mice. The involvement of the hormone leptin was tested by injection of recombinant protein vs. vehicle during the preconditioning period.

Results: CR-mediated protection was dose dependent up to 50% with maximal 2-fold effect sizes. PR benefits were abrogated by EAA re-addition and additive with CR, with maximal benefits at any given amount of CR occurring with a protein-free diet. GCN2 was not required for functional benefits of PR. Activation and repression of nutrient/energy-sensing kinases, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1), respectively, on PR reflected a state of negative energy balance, paralleled by 13% weight loss and an 87% decrease in leptin, independent of calorie intake. Recombinant leptin administration partially abrogated benefits of dietary preconditioning against renal IRI.

Conclusions: In male mice, PR and CR both contributed to the benefits of short-term DR against renal IRI independent of GCN2 but partially dependent on reduced circulating leptin and coincident with AMPK activation and mTORC1 repression.

Keywords: AMPK; GCN2; amino acid sensing; calorie restriction; dietary restriction; geometric framework; ischemia reperfusion injury; leptin; mTOR; protein restriction.

Conflict of interest statement

Author disclosures: LT Robertson, JH Treviño-Villarreal, P Mejia, Y Grondin, E Harputlugil, C Hine, D Vargas, H Zheng, CK Ozaki, BS Kristal, SJ Simpson, and JR Mitchell, no conflicts of interest.

© 2015 American Society for Nutrition.

Figures

FIGURE 1

Dose-dependent effects of 1 wk of CR against bilateral renal IRI in male mice in experiment 1. (A) Schematic of preconditioning and postconditioning feeding regimens relative to the timing of surgical renal IRI induction (arrow). (B) Schematic of diets with pie slices proportional to the calorie content of the indicated macronutrient and circle areas proportional to the indicated level of daily energy intake. (C) Total food intake during the preconditioning period of the indicated groups; n = 2–3 cages/group. (D) Mouse weights during the preconditioning period are expressed as percentage of starting weight. (E) Postoperative weight on day 3 after IRI is expressed as a percentage of change after IRI. (F) Kidney function after renal IRI is expressed as a percentage of serum urea AUC of the AL group. (G–I) Representative hematoxylin and eosin–stained kidney sections from the indicated diet groups 3 d after injury. Arrows point to areas of tubular necrosis as evidenced by nuclear changes and cytoplasmic lysis or vacuolization. Arrowheads show tubular cast formation. Asterisks are positioned over tubules that show a normal structural characteristic. Scale bar = 50 μm. (J) Histopathology scores indicative of renal damage with 1 representing the least (0–25%) tubular damage and 4 representing the most (75–100%). Values are means ± SEMs; n = 8–13 mice/group. *,**,***,****Different from the AL diet group: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. AL, ad libitum; CR, calorie restriction; G, glomerulus; IRI, ischemia reperfusion injury; post-op, postoperative; pre-op, preoperative.

FIGURE 2

Effects of 1 wk of protein vs. carbohydrate restriction against renal IRI in male mice in experiment 2. (A) Schematic of complete or restricted diets that lack calories specifically from protein (protein CR) or sucrose (sucrose CR) with pie slices proportional to the calorie content of the indicated macronutrient. (B) Total food intake during the preconditioning period of the indicated groups, n = 2–3 cages/group. (C) Mouse weights during the preconditioning period are expressed as percentage of starting weight. (D) Postoperative weight on day 3 after IRI is expressed as a percentage change after IRI. (E) Kidney function after renal IRI is expressed as a percentage of serum urea AUC of the complete group. Values are means ± SEMs; n = 8–14 mice/group. *,**,***,****Different from complete control diet group: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CR, calorie restriction; IRI, ischemia reperfusion injury; post-op, postoperative; pre-op, preoperative.

FIGURE 3

EAAs control benefits of 1 wk of PR against renal IRI in male mice in experiment 3. (A) Schematic of diets in which 18% of calories are contributed by protein (complete), sucrose (PF), NEAAs (NEAA only), or EEAs (EAA only); mice were preconditioned with these diets consumed AL (B–D) or restricted daily by ∼50% (E–G). (B, E) Daily food intake is expressed relative to mouse weight; n = 2 cages/group. (C, F) Mouse weights during the preconditioning period are expressed as percentage of starting weight. (D, G) Kidney function after renal IRI is expressed as a percentage of serum urea AUC of the complete diet group. Values are means ± SEMs; n = 4–5 mice/group. *,**,***,****Different from complete control diet group: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. AL, ad libitum; CR, calorie restriction; EAA, essential amino acid; IRI, ischemia reperfusion injury; NEAA, nonessential amino acid; PF, protein free; PR, protein restriction; pre-op, preoperative.

FIGURE 4

Effects of 1 wk of combined protein restriction and CR against renal IRI in male mice in experiment 4. (A) Total food intake is expressed relative to body weight with the indicated diet, with PF groups at the indicated daily energy intake and the AL complete diet group from Figure 1 as a reference; n = 2–3 cages/group. (B) Mouse weights during the preconditioning period are expressed as percentage of starting weight. (C) Postoperative weight on day 3 after IRI is expressed as a percentage of change after IRI. (D) Kidney function after renal IRI is expressed as a percentage of serum urea AUC of the AL complete diet group. Values are means ± SEMs; n = 9–13 mice/group. *,**,***,****Different from complete control diet group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Exponential fit of observed AUC serum urea data calculated as a percentage of the AL complete group in the corresponding experiment relative to total energy intake and expressed as percentage of calorie restriction. Observed AUC serum urea for the complete diet groups (circles) was best fitted by the equation AUC serum urea = 99 × exp(−0.0125 × CR) with strong positive Pearson’s correlation (r = 0.83, P < 0.001) between the observed and predicted data. The observed AUC serum urea for the PF diet groups (triangles) was best fitted by the equation AUC serum urea = 73 × exp(−0.0114 × CR), also with a strong Pearson’s correlation (r = 0.81, P = 0.029). (F) Geometric framework-based model of the nutritional basis of short-term dietary restriction. Axes represent daily energy intake of protein (x) or carbohydrates and fat (y). Colors indicate functional outcome of renal IRI as a percentage of dysfunction in mice that consumed a complete diet AL, with dark red indicating maximal dysfunction and dark blue maximal protection. Each dotted line represents a continuum of isocaloric diets that differ in macronutrient ratios; solid vertical lines represent diets with 0%, 18%, or 36% calories from protein as indicated. AL, ad libitum; Comp, complete diet; CR, calorie restriction; IRI, ischemia reperfusion injury; PF, protein free; post-op, postoperative; pre-op, preoperative; prot, protein.

FIGURE 5

Protein- and energy-sensing signal transduction in kidney extracts from male mice after 1 wk of protein and/or CR in experiment 5. (A) Immunoblots of markers of GCN2 activation in kidney extracts from mice fed the indicated Comp or PF diet consumed AL or restricted ∼50% (CR). (B) Serum urea on day 1 after renal IRI of WT and GCN2 KO mice preconditioned with the indicated diet 1 wk before surgery in experiment 6; n = 5–9 mice/group. **Different from complete control within genotype, P < 0.01. (C) Immunoblots of mTORC1 target S6K, AMPK, and AMPK target TSC2 in kidney extracts from mice fed the indicated diets. (D–F) Quantitation of p/t ratios of S6K pThr389 (D), AMPK pThr172 (E), TSC2 pThr1387 (F); n = 3–5 mice/group. Main effects of dietary protein (protein = Comp vs. PF), food restriction (calories = AL vs. CR), and interactions are indicated above each graph with P values; ns, P > 0.05. Means without a common letter differ, P < 0.05. Values are means ± SEMs. AL, ad libitum; AMPK, AMP-activated protein kinase; ATF4, activating transcription factor 4; Comp, complete diet; CR, calorie restriction; eIF2α, eukaryotic initiation factor 2α GCN2, general control non-derepressible 2; IRI, ischemia reperfusion injury; KO, knockout; PF, protein-free diet; p/t, phospho/total; S6K, p70 ribosomal S6 protein kinase; TSC2, tuberous sclerosis complex 2; WT, wild-type.

FIGURE 6

Metabolic and serum variables of male mice after 1 wk of protein and/or CR in experiment 5. (A, B) Combined means of individual serum EAAs (A) and NEAAs (B) calculated as a percentage of the AL complete diet group, after 1 wk fed the indicated diet. (C) Body weight is expressed as a percentage of the starting value. (D) Total food intake during the preconditioning period of the indicated groups; n = 2 cages/group. (E, F) Serum concentrations of adiponectin (E) and leptin (F) after 1 wk fed the indicated diet. Main effects of dietary protein (protein = complete vs. PF), food restriction (calories = AL vs. CR), and interactions are indicated above each graph with P values; ns, P > 0.05. Means without a common letter differ, P < 0.05; n = 5 mice/group. (G) Serum urea on day 1 after renal IRI of mice preconditioned with the indicated diet 1 wk before surgery and treated with leptin or vehicle in experiment 7 as indicated. **Different from vehicle control within diet, P < 0.01; n = 10–15 mice/group. Values are means ± SEMs. AL, ad libitum; Comp, complete diet; CR, ∼50% calorie restriction; EAA, essential amino acids; IRI, ischemia reperfusion injury; KO, knockout; NEAA, nonessential amino acids; PF, protein-free diet; WT, wild-type.