Dietary fat composition influences glomerular and proximal convoluted tubule cell structure and autophagic processes in kidneys from calorie-restricted mice

Abstract

Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age-related diseases in a wide range of animals, including non-human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age-related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.

Keywords: aging; calorie restriction; dietary fat; kidney; mice.

Figure 1

Protein expression levels of p16. Panel A shows CON and CRS mice (^a P < 0.05 vs CON after 6 months of dietary intervention) and panel B the three CR diet groups (*P < 0.5). In all figures, white bars refer to 6 and black bars to 18 months of dietary intervention. In panels A and B, two representative Western blot bands for each experimental group are shown.

Figure 2

Analysis of structural and ultrastructural features of renal glomeruli in CON and CR mice. Panels A to D show light microscopy pictures of glomerular structure after 6 (A, CON; B, CRS) and 18 months of intervention (C, CON and D, CRS). Panels E and F show examples of ultrastructural modifications of glomerular basement membrane (GBM), filtration slits (FS) (white arrows), and podocyte foot processes (asterisks) width after 18 months of intervention (E, CRS and F, CRF). In panel E, we show three examples of how measurements of GBM thickness were taken (two‐headed arrows). The results of quantification are included in panels G (GBM), H (FS), and I (PFP). Aging induced striking changes in all of these structures (**P < 0.01 and ***P < 0.001) when comparing 6 vs 18 months in the same diet group. In panel G, ^a P < 0.001 vs CON and CRL; ^b P < 0.001 vs CRL after 6 months of intervention; ^c P < 0.001 vs CON and CRL and ^d P < 0.001 vs CRL after 18 months of intervention. In panel H, ^a P < 0.001 vs all the other 6‐month‐old groups; ^b P < 0.001 vs all other 18‐month‐old groups; ^c P < 0.001 vs CRL and CRS in 18‐month‐old animals. In panel I, ^a P < 0.001 vs CRS after 6 months of intervention; ^b P < 0.001 vs CRS after 18 months of CR, and ^c P < 0.001 vs CRS and CRL after 18 months of CR.

Figure 3

Ultrastructural features of PCT epithelial cells (A) and nuclei (B), and mitochondrial planimetric (C and D) and stereological analysis (E and F) in PCT cells from the different dietary groups. In all panels *P < 0.05; **P < 0.01, and ***P < 0.001. In panel A, ^a P < 0.01 and 0.05 vs CRL and CRS in 6‐month‐old mice, respectively, and ^b P < 0.001 and 0.05 vs CRL and CRS in 18‐month‐old mice, respectively. In panel B, ^a P < 0.05 vs CRF after 6 months of intervention. Mitochondrial volume (C) and circularity coefficient (D) changed depending on age and dietary fat in CR groups. In panel C, ^a P < 0.001 vs CRS 6 months; ^b P < 0.001 and P < 0.01 vs CRS and CRF, respectively, after 6 months of CR;^c P < 0.001 vs CRS and CRF 18 months; ^d P < 0.001 vs CRF six months and ^e P < 0.001 vs CRF 18‐month‐old group (# denotes a linear trend CRL < CRS < CRF in 18‐month‐old animals; P < 0.001). In panel D, ^a P < 0.001 vs CON and CRL;^b P < 0.001 vs CRS and ^c P < 0.001 vs CRS and CRL in 18‐month mice (# denotes a linear trend of increased mitochondrial circularity coefficient CRL < CRS < CRF). The stereological parameters Vv and Nv are represented in E and F, respectively. In E, ^a P < 0.01 vs CRL and CON in 18‐month‐old animals and ^b P < 0.01 vs CRL in 6‐month‐old mice. In panel F, ^a P < 0.01 vs CON at 6 months of intervention and ^b P < 0.01 and P < 0.0 5 vs CRL and CRS, respectively, in 18‐month‐old animals. In panel F, # denotes a positive linear trend (P < 0.001) of decreasing Nv in calorie‐restricted animals for 18 months (CRL > CRS > CRF).

Figure 4

Representation of PGC1‐α (panels 4A and 4B), TFAM (panels 4C and 4D), and NRF1 (panels 4 E and 4F) protein expression levels in the different dietary groups during aging (*P < 0.05, **P < 0.01, and ***P < 0.001). In panel B, ^ap < 0.05 vs CRL and ^b P < 0.01 vs CRL and P < 0.05 vs CRS in 18‐month‐old groups. In panel C, ^a P < 0.05 vs CON. In panel D, ^a P < 0.05 vs CRL after 6 months of CR and ^b P < 0.05 vs CRL and CRF in 18‐month CR mice. A decreasing linear trend (^# P < 0.01) was found in old CR animals CR (CRL > CRS > CRF) for PGC1‐α, TFAM, and NRF1 expression levels. Two representative Western blot bands for each experimental group are also shown.

Figure 5

Ultrastructural localization of autophagic figures (arrows) in 18‐month control or CR mice (A, C, and D = CON; B and F = RCL; E = CRS; and G = RCF). Pictures A and B are podocytes from CON and CRL groups, respectively. Proximal convoluted tubular cells also showed autophagic figures regardless the dietary fat (Panels D, E, F, and G). In CON mice (panel C), a relatively high number of PCT showed an elevated number of enlarged lysosomes with characteristic concentric lamellar inclusions (asterisks). The results of a quantification of number of autophagic event figures in relation to cell area are shown in panel H (^a P < 0.05 vs CRS 18 months; ^b P < 0.01 vs CRS and CRF in 18‐month mice;^c P < 0.05 vs CRF in six‐month intervention; ^d P < 0.05 vs CRF 18 months). A positive linear trend of decreasing autophagic events in calorie‐restricted animals for 18 months (CRL > CRS > CRF) was also found (^# P < 0.001).

Figure 6

Representation of Beclin‐1 expression levels (Panels A and B) and LC3 ratio (LC3‐II/LC3‐II + LC3‐I; panels C and D) in the dietary groups. In panels A and C, we represent young and old controls and CR animals with soybean as dietary fat for Beclin‐1 (*P < 0.05) and LC3 ratio, respectively (^a,b P < 0.05 vs respective CON group). In panel B and D, we represent the effect of 6 and 18 months of CR with the different fat sources on proteins expression levels (Panel B: ^a P < 0.05 vs RCS and *P < 0.05; panel D: ^a,b P < 0.05 vs respective CRS animals). In all panels, two representative Western blot bands for each experimental group are shown.