AJP - Regu Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 293: R1259-R1266, 2007. First published June 20, 2007; doi:10.1152/ajpregu.00231.2007
0363-6119/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/R1259    most recent
00231.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tarry-Adkins, J. L.
Right arrow Articles by Ozanne, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tarry-Adkins, J. L.
Right arrow Articles by Ozanne, S. E.

DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Protein restriction in lactation confers nephroprotective effects in the male rat and is associated with increased antioxidant expression

Jane L. Tarry-Adkins,1 Jaap A. Joles,2 Jian-Hua Chen,1 Malgorzata S. Martin-Gronert,1 Dionne M. van der Giezen,3 Roel Goldschmeding,3 C. Nicholas Hales,1,{dagger} and Susan E. Ozanne1

1Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom; and 2Departments of Nephrology and Hypertension and 3Pathology, University Medical Centre, Utrecht, The Netherlands

Submitted 5 April 2007 ; accepted in final form 14 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Telomere shortening has been implicated in the aging process and various age-associated disorders, including renal disease. Moreover, oxidative stress has been identified as an initiator of accelerated telomere shortening. We have shown previously that maternal protein restriction during lactation leads to reduced renal telomere shortening, reduced albuminuria, and increased longevity in rats. Here we address the hypothesis that maternal protein restriction during lactation is nephroprotective and associated with increased expression of antioxidative enzymes and decreased age-dependent renal telomere shortening. Newborn rats were suckled by a dam fed either a control (20% protein) or low-protein (8% protein) diet. All animals were weaned onto standard chow. Offspring that had been suckled by protein-restricted mothers had reduced albuminuria, N-acetyl-glucosaminidase, and urinary aldosterone excretion. These animals also did not show significant age-dependent renal telomere shortening and hence had significantly longer telomeres at 12 mo of age. This lack of renal telomere shortening was associated with increased levels of the antioxidant enzymes manganese superoxide dismutase, glutathione peroxidase, and glutathione reductase. These findings suggest that beneficial effects of slow growth during lactation are associated with increased antioxidant capacity and prevention of age-dependent telomere shortening in the kidney.

renal disease; albuminuria; telomeres; antioxidant enzymes

PREVALENCE OF CHRONIC renal disease (CRD) and resultant end stage renal failure is threatening to reach epidemic proportions over the next decade (30) with CRD rates growing at 8% per annum (1). Many strategies have been shown to prevent progression of renal disease, including the control of proteinuria (1). In cases of renal disease, postnatal protein restriction has been demonstrated to be effective in nephroprotection in both adult rats (4, 20) and humans (14). However, the possible renoprotective effects of protein restriction during specific time periods, such as lactation, are not clear. In our laboratory, a rat model of protein restriction during lactation, whereby rat pups born to dams fed a 20% protein diet were cross-fostered to 8% protein-fed mothers until weaning, produced offspring with a normal birth weight that grew slowly during lactation (22). When weaned onto standard laboratory chow, these animals had significantly increased longevity (22) and reduced albuminuria throughout life (33) compared with the control offspring of mothers fed a 20% protein diet during pregnancy and lactation.

Beneficial effects of reduced nutrition and, consequently, slow growth during lactation have been demonstrated in human studies. These have primarily been observed in studies comparing the long-term effects of formula feeding compared with breast-feeding. The latter, in general, is associated with a lower plane of nutrition and, consequently, slower growth during suckling. Studies have also shown that formula-fed infants are more overweight later in childhood than breast-fed infants (18). Studies of premature babies randomly fed infant feeding formula or breast milk have also demonstrated that those that were breast-fed had reduced atherosclerotic risk factors later in childhood (38). Moreover, slower growth during the first 2 wk of life has been suggested to be particularly beneficial in this low-birth-weight population (39).

The mechanisms by which nutrition and growth during the suckling period impact on long-term health and longevity is unknown. However, our previous studies have suggested that effects on the rate of telomere shortening may play a role (22, 23). Telomeres have been implicated in the aging process for a number of years (2), and it has been suggested that telomere shortening may be linked to age-associated diseases, including kidney dysfunction (17). Oxidative stress has been implicated in accelerated telomere shortening, as oxidative damage is less well repaired in telomeric DNA than elsewhere in the chromosome (36, 46). Three major antioxidant enzymes in the oxidative stress defense network are manganese superoxide dismutase (MnSOD), glutathione peroxidase (Gpx). and glutathione reductase (GR). MnSOD is located predominately in the mitochondria and is essential for life as demonstrated by the neonatal lethality of MnSOD-deficient mice (27). In addition, mice expressing 50% normal levels of MnSOD show increased susceptibility to oxidative stress and severe mitochondrial dysfunction due to increased reactive oxidative species (27). However, the association between MnSOD, increased oxidative stress, and lifespan has been not shown in all studies (44). Moreover, MnSOD overexpression has been shown to be protective in cisplatin-induced renal injury (9). Gpx is particularly important in end stage renal disease with significant reduction of this antioxidant enzyme reported in patients with diabetic nephropathy and chronic renal failure and in those undergoing hemodialysis (7, 13, 26). GR-deficient mice subjected to reactive oxidative species-mediated injury demonstrated renal proximal tubule damage (35).

The hypothesis of this study was therefore that the nephroprotective effects of protein restriction in lactation are associated with reduced renal telomere shortening and increased protein expression of three major antioxidant enzymes: MnSOD, Gpx-1, and GR. Markers of renal dysfunction were measured [including albuminuria, a marker of glomerular damage and an established risk marker for progressive renal functional loss (11, 31)] and N-acetyl-glucosaminidase (NAG) excretion [a marker of proximal renal tubule damage (8, 16)]. Aldosterone, a potential mediator of renal injury was also measured (12, 15, 25). Creatinine excretion and clearance were also analyzed. Renal histological assessment was conducted in the same animals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

All of the procedures involving animals were conducted under the British Animals (Scientific Procedures) Act (1986) under license (PIL80/8594). Wistar rat dams were placed on an ad libitum standard laboratory chow diet (20% protein) and water until pregnancy was confirmed through observation of vaginal plugs. When pregnancy was confirmed, dams were fed either a 20% normal protein isocaloric diet (control) or an isocaloric low-protein (LP) (8%) diet. Both of these diets were purchased from Arie Blok, Woerden, The Netherlands, and the detailed dietary composition has been previously described (40). Cross-fostering techniques were then used to generate protein-restricted offspring during lactation. The postnatal protein restriction (PLP) group was offspring of rat dams fed the control diet during pregnancy and then nursed by rat dams fed an LP (8%) diet. The control group was control-fed offspring that were suckled by the same control-fed rat dams. To prevent any stress or rejection to the animals involved in cross-fostering, the pups were transferred with some of their own bedding. No pup rejection or stress was observed following this procedure. All animals were weaned onto a standard diet containing 20% protein (SDS, Witton, UK) from 21 days of age and remained on it until the end of the study. Mean litter weights were recorded between days 3 and 21.

Because we have recently documented accelerated telomere shortening and more albuminuria in male than in female rats (42), the present study was performed in males. One male from each litter was maintained until 3 mo of age and then killed. Immediately after death, the kidneys were removed. The left kidney was visually dissected into cortex and medulla. Tissue was snap frozen in liquid nitrogen and stored at –80°C until required for analysis. A second male from each litter was maintained and was used for protein and DNA analysis, until 12 mo of age when blood, left kidney (cortex and medulla), and the whole right kidney was collected for histology. Twenty-four hour urine samples were also taken at 1, 3, 5, 7, and 9, and 12 mo of age. During urine collection, rats were housed in metabolic cages with free access to chow and water for 24 h.

Telomere Length Measurement

Nonsheared, high-molecular size DNA (average size 97 kb) was isolated from the cortex and medulla by using a commercial kit for DNA extraction using the protocol provided by the manufacturer (Qiagen). DNA quantity and integrity were determined using a spectrophotometer (GeneQuant; Pharmacia Biotech). DNA (1.2 µg) was digested with HinfI and RsaI restriction enzymes (6, 42). The digested DNA was then separated using pulsed-field gel electrophoresis (6, 42). After electrophoresis, the gels were checked for nonspecific degradation of the undigested DNA and complete digestion of the digested DNA by staining with ethidium bromide. The gels were visualized using an Alpha Imager UV light source (Alpha Innotech) and photographed with a P/N Polaroid film. The separated DNA fragments were transferred by Southern blot analysis onto nylon-positive membranes (Roche Diagnostics), and telomeric repeat length was determined using a slightly modified commercial method of chemiluminescent detection (42). The telomere signals were analyzed using Adobe Photoshop and MacBas computer software. Molecular weight markers used per gel were a midrange pulsed-field gel marker (New England Biolabs), and dioxygenin (low-range) molecular weight marker (Roche Diagnostics). Standard undigested and digested genomic control DNA was run on each gel to demonstrate the efficiency of the digestion and to minimize any intergel differences. Gels were run in duplicate to produce average %telomere length values. Each gel was accepted on the criteria that the %telomere length of the control DNA in any of the four telomeric regions analyzed (1.3–4.2 kb, 4.2–8.6 kb, 8.6–48.5 kb, and 48.5–145 kb) was < 1.5 SD from the mean.

Telomere length was measured as described previously, whereby the percentage of intensity (%telomere length) of the telomeric signal was determined in four molecular size regions as defined by molecular weight markers. Specific grid squares were placed around the telomeric smear according to the previously detailed molecular weight regions. %Telomere length (expressed as %photo-stimulated luminescence) was measured (6, 42).

Antioxidant Enzyme Quantification

Western blot analysis was used to determine expression levels of MnSOD, Gpx-1, and GR in the cortex and medulla of the same animals. Protein was extracted by hand homogenization of 1 mg of renal tissue in 1 ml TK lysis buffer [50 mM HEPES, 150 mM NaCl, 20% Triton X-100, 100 mM Na Orthovanadate, 500 mM NaF, 100 mM Na4P2O7, 500 mM EDTA (pH8)], and the resultant supernatant was then assayed by using copper sulfate and bicinchoninic acid (Sigma-Aldrich, St. Louis, MO). Protein (20 µg) was loaded onto 10% polyacrylamide gels, electrophoresed, and transferred to polyvinylidene difluoride membrane (42). MnSOD was detected using anti-MnSOD (type II)-specific rabbit IgG (Upstate Biochemicals), Gpx-1 was detected using anti-Gpx-1-specific rabbit IgG (Ab-cam), and an anti-GR-specific rabbit IgG (Abcam) was used to measure GR. To check linearity and reproducibility, a standardization gel consisting of one sample (S1–20 µg protein), 50% of the same sample S1 (50%), and a further 20 µg protein from a different sample (S2) were loaded five times onto one gel. Each gel was then loaded with 20 µg protein per sample plus the same S1, S1 (50%), and S2 samples. Gels were accepted on the criterion that the ratios of S1/S1 (50%) and S1/S2 were less than the mean integrated density value (arbitrary units) of the standardization gel ± 1.5 SD.

Urine and Serum Analysis

Albuminuria was measured by using a rat-specific enzyme-linked immunoassay (IDS; Spi-Bio). Results were expressed as milligram per day. NAG activity was measured by using a colorimetric method (PPR Diagnostics). Results were expressed as micromole per day. Urine and serum aldosterone were measured by using a goat anti-mouse IgG immunoassay kit (Cayman Chemicals). Results were expressed as nanograms per day and picograms per day, respectively. Urine and plasma creatinine were measured by the Jaffé method (Sigma, St. Louis, MO).

Histological Assessment

The formalin-fixed right kidneys from 12-mo-old rats were embedded in paraffin blocks and microtomed into 4-µm-thick sections, stained with PAS and blindly analyzed by an independent histopathologist. Fifty glomeruli were scored per rat for proliferation, matrix expansion, sclerosis, and adhesion in quadrants with 0 indicating no change and 4 indicating a change in all quadrants. A score of 1 or 2 was regarded as partial focal glomerulosclerosis (FGS) and a score of 3 or 4 as total FGS. Incidence of FGS (partial or total) is expressed as a percentage.

Statistical Analysis

Telomere length, MnSOD levels, and aldosterone excretion were analyzed by using a factorial two-way ANOVA with maternal diet and age as the independent variables, followed by Duncan's post hoc testing where appropriate. Albumin and NAG excretions were analyzed using a repeated-measures two-way ANOVA with maternal diet and age as the independent variables. Student-Newman-Keuls post hoc testing was used for the albumin and NAG excretion data. All unpaired data (histological assessment) were analyzed using Student's t-test. All data were represented as mean ± SE, unless stated otherwise. Albuminuria and NAG excretion were represented as log-transformed data. In all cases, a P value < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physical Characteristics

Average litter weights and sizes and body weights. No significant difference in litter weight at birth or litter size was seen between groups (data not shown). The PLP group, which had birth weights similar to the control animals, became significantly (P < 0.001) smaller than the control group by day 7 and remained smaller throughout lactation (Table 1). These differences remained with the PLP animals being significantly (P < 0.001) lighter than controls until the end of the study (Fig. 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Effect of lactation protein restriction on preweaning body weights of male rats

 

Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 1. Effect of lactation protein restriction on postweaning body weights of male rats (n = 15 per group). PLP, postnatal protein restriction. Data were collected (after 24-h urine collection) at 1, 3, 5, 7, 9, and 12 mo of age. Means ± SE. *P < 0.05, ***P < 0.001 PLP vs. control.

 
Kidney weights. The PLP animals showed significantly (P < 0.001) reduced absolute kidney weights at both 3 and 12 mo of age. However, relative kidney weights remained similar between groups at both ages (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Effect of lactation protein restriction on absolute (g) and relative kidney weights at 3 and 12 mo of age

 
Urine and Serum Analysis

Albuminuria. The PLP animals were significantly less albuminuric compared with the control group at 5, 7, 9, and 12 mo of age (P < 0.05 to P < 0.01) (Fig. 2A). Urinary albumin excretion significantly (P < 0.001) increased with age in both groups.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Effect of lactation protein restriction on 24-h urinary albumin excretion (A) and N-acetyl-glucosaminidase (NAG) excretion (B) in 1-, 3-, 5-, 7-, 9-, and 12-mo-old male rats (n = 12 per group). *P < 0.05, **P < 0.01, ***P < 0.001 PLP vs. control, ###P < 0.001 1- vs. 3-mo-old control and PLP rats.

 
Urinary NAG excretion. Significantly (P < 0.001) reduced levels of urinary NAG were found in PLP compared with control at 1 mo of age (Fig. 2B). An initial significant (P < 0.001) rise in urinary NAG excretion was observed between 1 and 3 mo of age in both groups.

Serum aldosterone concentration and urinary aldosterone excretion. There was a significant effect of maternal diet on urinary aldosterone (P < 0.05) with levels being reduced in the PLP group (Fig. 3A). There was also a significant effect of age (P < 0.001) with levels increasing with age in both groups. There was a significant interaction between maternal diet and age (P = 0.041) with urinary aldosterone increasing more with age in the control animals. At both 3 and 12 mo of age no significant difference in serum aldosterone was observed between groups (Fig. 3B), and no significant difference in serum aldosterone was seen between 3 and 12 mo of age.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Effect of lactation protein restriction on urinary aldosterone (A) and serum aldosterone (B) excretion. Measurements were made in 3- and 12-mo-old male rats (n = 12 per group). *P < 0.05 PLP vs. control, ###P < 0.001 3- vs. 12-mo-old control rats, and ##P < 0.01 3- vs. 12-mo-old PLP rats.

 
Creatinine parameters. At 12 mo of age there were no significant differences in creatinine excretion (corrected for body weight) (18 ± 4 µmol·100 g body wt–1·24 h–1 vs. 25 ± 2 µmol·100 g body wt–1·24 h–1), plasma creatinine (28 ± 3 µmol/l vs. 31 ± 2 µmol/l) or creatinine clearance (971 ± 228 µl·100 g body wt–1·min–1 vs. 1,174 ± 75 µl·100 g body wt–1·min–1 between control and PLP groups, respectively).

Telomere Length Data

Effect of maternal diet. At 3 mo of age, no significant difference in kidney cortical telomere length was seen between any groups. However, by 12 mo, significantly (P < 0.05) fewer short telomeres (1.3- to 4.2-kb size range) were observed in the PLP group compared with the control group in the cortical region (Fig. 4A). No difference in telomere length was observed in the medulla region at 3 or 12 mo of age (Fig. 4B).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 4. Effect of lactation protein restriction on telomere length in 3- and 12-mo-old male rats, in cortex (A) and medulla (B; n = 8 in all groups). *P < 0.05 PLP vs. control. #P < 0.05, ##P < 0.01 3- vs. 12-mo-old control rats.

 
Effect of age. Significantly (P < 0.05) more short (1.3–4.2 kb; 21.6 ± 2.2% vs. 15.8 ± 2.1%) and significantly (P < 0.01) fewer long (48.5–145 kb; 17.9 ± 1.5% vs. 24.2 ± 1.5%) telomeres were found in the kidney cortex of control animals at 12 mo compared with 3 mo. In contrast, no significant difference in telomere length was observed in the cortical region of the PLP group at 12 mo compared with 3 mo (48.5–145 kb; 20.4 ± 1.3% vs. 23.5 ± 2.8%) and (1.3–4.2 kb; 14.9 ± 1.9% vs. 11.3 ± 2.3%) 12 mo compared with 3 mo (Fig. 4A). In the medulla, no significant difference in telomere length was observed at 12 mo compared with 3 mo (Fig. 4B).

Antioxidant Enzyme Data

All protein expression data are expressed as a percentage of the mean value of 3-mo cortex or medulla samples as appropriate.

MnSOD. No significant difference in MnSOD protein expression levels were observed at 3 mo of age in either cortex or medulla regions between the two groups. However, by 12 mo, significantly (P < 0.05) more MnSOD protein was detected in the cortex of the PLP group compared with controls (Fig. 5A). In the medulla, at 12 mo of age no significant difference in MnSOD levels was observed (data not shown). There was no effect of age on MnSOD expression.


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 5. Effect of lactation protein restriction on manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (Gpx-1), and glutathione reductase (GR) protein expression levels in the cortex of 3- and 12-mo-old male rats (n = 8 in all groups). *P < 0.05, **P < 0.01 PLP vs. control, #P < 0.05, and ##P < 0.01 3- vs. 12-mo-old control rats.

 
Gpx-1 and GR. At 3 mo, significantly (P < 0.01) increased Gpx-1 and GR protein expression levels were observed in the cortex of the PLP group compared with controls; however, by 12 mo, levels of Gpx-1 and GR were similar between groups (Figs. 5, B and C). In the medulla region, no significant difference at either age was seen between control and PLP groups (data not shown). There was a significant interaction between maternal diet and age with a significant upregulation of Gpx-1 and GR in the control group between 3 and 12 mo; however, Gpx-1 and GR levels in the PLP group remained stable between ages.

Histology of the Renal Cortex

Scoring for FGS revealed no statistical differences in either partial (11.5 ± 2.0% vs. 8.5 ± 1.1%) or total FGS (9.5 ± 2.3% vs. 4.9 ± 1.0%) between control and PLP groups, respectively. Representative sections are shown in Fig. 6. Histological analysis was not performed on the renal medulla due to sample insufficiency.


Figure 6
View larger version (147K):
[in this window]
[in a new window]

  		Fig. 6. Effect of lactation protein restriction on histology of renal cortical tissue from 12-mo-old male rats. Representative sections are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of this study was to investigate nephroprotective effects of protein restriction during lactation in male Wistar rats and to explore associated mechanisms. The kidney is a major organ of interest in the rat, as it has been reported that the most common cause of death among laboratory rats is renal failure (21). We have previously shown that male rats that were protein restricted during lactation lived significantly longer than controls (22) and had significantly reduced albuminuria over aging (33). We explored the potential mechanisms of this by examining telomere length, renal function, and antioxidative stress enzymes.

Consistent with previous findings, PLP offspring were of similar birth weight to controls but grew slowly during lactation so that they were significantly smaller than the controls by day 7. These body weight trajectories were maintained throughout the study. In absolute terms, the PLP group demonstrated significantly smaller kidneys at both 3 and 12 mo; however, there was no significant change in renal weight when expressed relative to body weight.

No effect of maternal diet was seen in renal telomere length at 3 mo of age. By 12 mo of age, significantly fewer short (1.3–4.2 kb) cortical telomeres were observed in the PLP group compared with controls. Significant difference in the smallest telomere length may be particularly relevant, as it has been shown that the shortest telomere, not the average telomere length, is critical for cell viability and chromosome stability in telomerase null mice (19). The significant effect of maternal diet on telomere length was only observed in the cortex region of the kidney and not in the medulla. This interesting regional disparity suggests that cortical telomeres may be more susceptible to the effects of maternal diet than those in the medulla. Telomere shortening with age has also been shown to be greater in the kidney cortex than the medulla in both humans (29) and rats (42). This may be linked to differences in mitochondrial density (28) and, consequently, oxidative damage between the two regions.

It is well known that oxidative stress can accelerate telomere shortening (24, 46), and, as the kidney is one of the most metabolically active of all tissues (43), we measured levels of three major antioxidant enzymes in both renal regions at 3 and 12 mo of age. MnSOD is a mitochondrial antioxidant enzyme responsible for the dismutation of O·2– into H2O2. Gpx-1 and GR are integral components of the thioredox system (43). At 3 mo, significant upregulation of cortical Gpx-1 was seen in the PLP group, suggesting that the PLP group could be more efficient in catalyzing H2O2 into H2O and O2. Moreover, significant upregulation of cortical GR in the PLP group demonstrates that the PLP group is able to provide more glutathione substrate for Gpx-1 to utilize. This is consistent with the idea that low-intensity biological stress on an organism elicits a defense response that helps protect it against the causes of aging. This hormesis hypothesis has been proposed previously to explain the beneficial effects of caloric restriction on aging (34, 37). The current data suggest that a similar response may occur in response to maternal protein restriction during the suckling period. By 12 mo of age, no significant difference in either of these antioxidant enzymes was seen between groups.

At 3 mo, MnSOD levels between the groups were not significantly different; however, by 12 mo, cortical levels of MnSOD were significantly elevated in the PLP group compared with controls. This suggests that the PLP group is able to upregulate MnSOD protein expression in response to O·2–, which accumulates with age; conversely the control group was unable to facilitate this upregulation of MnSOD. This suggests enhanced mitochondrial antioxidant protection in the PLP group at 12 mo, as MnSOD is primarily present in mitochondria. Moreover, downregulation of MnSOD has been demonstrated in rats with chronic renal failure, which was accompanied by oxidative stress (45), and mice expressing 50% normal MnSOD levels show increased susceptibility to oxidative stress and severe mitochondrial dysfunction (27). Moreover, MnSOD overexpression has been demonstrated to exert protective effects in cisplatin-induced renal injury (9). Taken together, these data may suggest that our PLP group is at a reduced risk of oxidative stress, chronic renal failure, renal injury, and mitochondrial dysfunction than the control group. Moreover, our data suggest the increased levels of these antioxidant enzymes may be acting to protect the kidney cortical telomeres of the PLP group from accelerated shortening. Indeed, this may partially explain why the PLP group had fewer short cortex telomeres compared with controls. Moreover, we observed that cortical telomeres significantly shortened with age in the controls; however, no significant age-related change in cortical telomere length was seen in the PLP group. This suggests that the PLP group may be protected from significant age-related cortex telomere shortening, perhaps by the upregulation of Gpx-1 and GR. Indeed, concentrations of Gpx have been shown to decrease in diabetic nephropathy, which may suggest that the increase in Gpx-1 in the PLP group is renoprotective (7). To our knowledge, this is the first time that upregulation of three major antioxidant enzymes in the kidney has been associated with protein restriction in lactation.

A range of markers of renal function were measured to investigate the effects of maternal diet on renal dysfunction. Urinary albumin excretion is an acknowledged method to measure renal glomerular damage and is an established marker of progressive renal functional loss (11, 31). Overall, albuminuria was lower in the PLP group, as we have previously demonstrated (33). Unexpectedly we found that NAG excretion, an established way of measuring proximal renal tubule damage (8), was strikingly lower in the PLP group at 1 mo of age. This early difference in NAG excretion is likely to reflect a loss of tubular function at an early stage in the control group, namely quantitative loss of the capacity of the proximal tubules to reabsorb proteins. This functional defect is no longer visible when the protein reabsorptive capacity is exceeded by more and more protein that has leaked through the glomeruli. Collectively, these findings suggest that tubular protein reabsorption is affected earlier by the standard diet than glomerular permselectivity.

It is established that aldosterone plays a critical role in many forms of renal disease (10), and oxidative stress is thought to play a key role in the development of aldosterone-induced renal injury. The reduced urinary aldosterone at both ages in PLP animals may be again indicative of these animals being subjected to less renal injury and oxidative stress. Because serum aldosterone was not significantly different between groups at either 3 or 12 mo of age, differences in urinary aldosterone excretion could be due to local synthesis of aldosterone in the kidney. Thus it is possible that the reduction of aldosterone excretion in the PLP group is related to renal protection. Overall renal function, as measured by plasma creatinine and creatinine clearance revealed no significant effect of maternal diet. We believe this is due to hyperfiltration of the nonsclerotic glomeruli in the control group, which is known to enhance albuminuria (41) and eventually lead to renal dysfunction. Indeed, the male Wistar rat strain was shown to develop slowly evolving glomerular injury by 19 mo of age (3), suggesting that at a later time point stronger differences in renal injury and kidney function may have become apparent.

In summary, these data suggest that the increased longevity of the postnatal growth-restricted offspring may be related to the absence of significant age-associated reduction of long cortical telomeres that were present in the control animals. This may partially be explained by the significant upregulation at 12 mo of cortical MnSOD that may act as a protective mechanism to preserve cortical renal telomere length in the older PLP animals. Increased levels of Gpx-1 and GR in the PLP group at 3 mo suggest that protein restriction during the suckling period exerts a mild oxidative stress to which Gpx-1 and GR are upregulated as a protective mechanism, akin to the idea of the hormesis hypothesis, recently proposed to explain the protective effects of caloric restriction (34, 37). Reduced levels of urinary aldosterone in the PLP group may also indicate that the PLP animals experience less renal damage with age as aldosterone has been demonstrated to play a key role in structural renal injury, extracellular matrix accumulation, and renal fibrosis (5, 25). In addition, aldosterone has been shown to stimulate oxidative stress (5) and an aldosterone antagonist has also been shown to increase expression of MnSOD, copper-zinc SOD, and catalase antioxidant enzymes, suggesting oxidative stress protection (32). It is also known that aldosterone can induce renal glomerular dysfunction leading to albuminuria (15). Throughout aging, the PLP animals have reduced albuminuria and NAG excretions compared with the control group, which may be reflective of the PLP animals experiencing less renal damage in terms of less glomerular and proximal renal tubule damage. The renoprotective effect of lowering protein intake in adult rats in the course of renal injury is well known (4, 20). However, such pronounced effects when restricted to the lactation period are a novel finding.

We therefore propose as a working hypothesis that reduced albuminuria and lower NAG excretion is reflective of reduced renal injury in terms of reduced glomerular and proximal renal tubule damage, suggesting that renoprotection may be present in the PLP group. The upregulation of MnSOD, Gpx-1, and GR antioxidant enzymes may also suggest that the PLP group confers oxidative stress protection. Moreover, the upregulation of antioxidant enzymes and fewer renal cortex telomeres may explain the observed increased longevity of rats, which grew slowly during the lactation period.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Parthenon Trust, The European Union Grant FOOD-CT-2005–007036, The British Heart Foundation, and National Institutes of Health.


   ACKNOWLEDGMENTS
 
We thank A. Wayman and A. Flack for their technical expertise.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Tarry-Adkins, Dept. of Clinical Biochemistry, Univ. of Cambridge, Addenbrookes Hospital, Box 232, Hills Rd., Cambridge, CB2 2QR, UK (e-mail: jane.adkins{at}ntlworld.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{dagger} Deceased September 15th, 2005. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alebiosu CO, Ayodele OE. The global burden of chronic renal disease and the way forward. Ethn Dis 15: 418–423, 2005.[Web of Science][Medline]
  2. Baird DM, Kipling D. The extent and significance of telomere loss with age. Ann NY Acad Sci 1019: 265–268, 2004.[CrossRef][Web of Science][Medline]
  3. Baylis C. Age-dependent glomerular damage in the rat. Dissociation between glomerular injury and both glomerular hypertension and hypertrophy. Male gender as a primary risk factor. J Clin Invest 94: 1823–1829, 1994.[Web of Science][Medline]
  4. Brenner BM, Meyer TW, Hostetter TH. Dietary protein intake and the progressive nature of kidney disease: the role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation and intrinsic renal disease. N Engl J Med 307: 652–659, 1982.[Web of Science][Medline]
  5. Brown NJ. Aldosterone and end-organ damage. Curr Opin Nephrol Hypertens 14: 235–241, 2005.[Web of Science][Medline]
  6. Cherif H, Tarry JL, Ozanne SE, Hales CN. Ageing and telomeres: a study into organ- and gender-specific telomere shortening. Nucleic Acids Res 31: 1576–1583, 2003.[Abstract/Free Full Text]
  7. Chiu YW, Kuo MC, Kuo HT, Chang JM, Guh JY, Lai YH, Chen HC. Alterations of glomerular and extracellular levels of glutathione peroxidase in patients and experimental rats with diabetic nephropathy. J Lab Clin Med 145: 181–186, 2005.[CrossRef][Web of Science][Medline]
  8. D'Amico G, Bazzi C. Urinary protein and enzyme excretion as markers of tubular damage. Curr Opin Nephrol Hypertens 12: 639–643, 2003.[CrossRef][Web of Science][Medline]
  9. Davis CA, Nick HS, Agarwal A. Mangenese superoxide dismutase attenuates Cisplatin-induced renal injury: importance of superoxide. J Am Nephrol 12: 2683–2690, 2001.
  10. Del Vecchio L, Procaccio M, Vigano S, Cusi D. Mechanisms of disease: the role of aldosterone in kidney damage and clinical benefits of its blockade. Nat Clin Pract Nephrol 3: 42–49, 2007.[CrossRef][Web of Science][Medline]
  11. De Zeeuw D, Remuzzi G, Parving HH, Keane WF, Zhang Z, Shahinfar S, Snapinn S, Cooper ME, Mitch WE, Brenner BM. Proteinuria, a target for renoprotection in patients with diabetic nephropathy: lessons from RENAAL. Kidney Int 65: 2309–2320, 2004.[CrossRef][Web of Science][Medline]
  12. Epstein M. Aldosterone blockade: an emerging strategy for abrogating progressive renal disease. Am J Med 119: 912–919, 2006.[CrossRef][Web of Science][Medline]
  13. Fellah H, Feki M, Souissi M, Ghorbel H, Ben Abdallah T, Massey Z, Hedhili A, Ben Maiz H, Lacour B, Kaabachi N, Mebezza A. Oxidative stress in end stage renal disease: evidence and association with cardiovascular events in Tunisian patients. Tunis Med 84: 728–729, 2006.
  14. Fouque D, Wang P, Laville M, Boissel JP. Low protein diet delays end-stage renal disease in non diabetic adults with chronic renal failure. Cochrane Database Syst Rev, CD001892, 2000.
  15. Greene EL, Kren S, Hostetter TH. Role of aldosterone in the remnant kidney model in the rat. J Clin Invest 98: 1063–1068, 1996.[Web of Science][Medline]
  16. Haas M, de Boer E, de Jong PE, Moolenaar F, Meijer DK, de Zeeuw D. Effects of anti-proteinuric therapy with angiotensin-converting-enzyme inhibition on renal protein catabolism in the adriamycin-induced nephrotic rat. Clin Sci (Lond) 105: 51–57, 2003.[Medline]
  17. Hales CN. Suicide of the nephron. Lancet 357: 136–137, 2001.[CrossRef][Web of Science][Medline]
  18. Harder T, Bergman R, Kallischnigg G, Plagemann A. Duration of breast-feeding and risk of overweight—a meta analysis. Am J Epidemiol 62: 397–403, 2005.
  19. Hemann MT, Strong MA, Hao LY, Greider CW. The shortest telomere, not the average telomere length is critical for cell viability and chromosome stability. Cell 107: 67–77, 2001.[CrossRef][Web of Science][Medline]
  20. Hutchison FN, Schambelan M, Kaysen GA. Modulation of albuminuria by dietary protein and converting enzyme inhibition. Am J Physiol Renal Fluid Electrolyte Physiol 253: F719–F725, 1987.[Abstract/Free Full Text]
  21. Iwasaki K, Gleiser CA, Masoro EJ, Masoro EJ, McMahon CA, Seo EJ, Yu BP. The influence of dietary-protein source on longevity and age-related disease processes in Fischer rats. J Gerontol 43: B5–B12, 1988.
  22. Jennings BJ, Ozanne SE, Dorling MW, Hales CN. Early growth determines longevity in male rats and maybe related to telomere shortening in the kidney. FEBS Lett 448: 4–8, 1999.[CrossRef][Web of Science][Medline]
  23. Jennings BJ, Ozanne SE, Hales CN. Nutrition, oxidative damage, telomere shortening and cellular senescence: individual or connected agents of aging? Mol Genet Metab 71: 32–42, 2000.[CrossRef][Web of Science][Medline]
  24. Kawanishi S, Oikawa S. Mechanism of telomere shortening by oxidative stress. Ann NY Acad Sci 1019: 278–284, 2004.[CrossRef][Web of Science][Medline]
  25. Lakkis J, Lu WX, Weir MR. RAAS escape: a real clinical entity that may be important in the progression of cardiovascular and renal disease. Curr Hypertens Rep 5: 408–417, 2003.[Web of Science][Medline]
  26. Loughrey CM, Young IS, Lightbody JH, McMaster D, McNamee PT, Trimble ER. Oxidative stress in haemodialysis. QJM 87: 679–683, 1994.[Abstract/Free Full Text]
  27. Macmillan-Crow LA, Cruithirds DL. Invited review: manganese superoxide dismutase in disease. Free Radic Res 34: 325–336, 2001.[Web of Science][Medline]
  28. Maunsbach AB, Christensen EI. Functional ultrastructure of the proximal tubule. In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, sect. 8, vol. I, chapt. 2, p. 41–108.
  29. Melk A, Ramassar V, Helms LM, Moore R, Rayner D, Solez K, Halloran PF. Telomere shortening in kidneys with age. J Am Soc Nephrol 11: 444–453, 2000.[Abstract/Free Full Text]
  30. Melquid El Nahas A, Bello AK. Chronic kidney disease: the global challenge. Lancet 365: 331–340, 2005.[Web of Science][Medline]
  31. Mogensen CE, Hansen KW, Osterby R, Damsgaard EM. Blood pressure elevation vs. abnormal albuminuria in the genesis and prediction of renal disease in diabetes. Diabetes Care 15: 1192–1204, 1992.[Abstract]
  32. Onozato ML, Tojo A, Kobayashi N, Goto A, Matsuoka H, Fujita T. Dual blockade of aldosterone and angiotensin II additively suppresses TGF-beta and NADPH oxidase in the hypertensive kidney. Nephrol Dial Transplant 22: 1314–1322, 2007.[Abstract/Free Full Text]
  33. Petry CJ, Jennings BJ, James LA, Hales CN, Ozanne SE. Suckling a protein-restricted dam leads to a diminished albuminuria in her male offspring in adult life: a longitudinal study. BMC Nephrology 7: 7–14, 2006.[CrossRef][Medline]
  34. Rattan SIS. Aging, anti-aging and hormesis. Mech Ageing Dev 125: 285–289, 2004.[CrossRef][Web of Science][Medline]
  35. Rogers LK, Bates CM, Welty SE, Smith CV. Diquat induces renal proximal tubule injury in glutathione reductase-deficient mice. Toxicol Appl Pharmacol 217: 289–298, 2006.[CrossRef][Web of Science][Medline]
  36. Saretzki G, von Zglinicki T. Replicative aging, telomeres and oxidative stress. Ann NY Acad Sci 959: 24–29, 2002.[Web of Science][Medline]
  37. Sinclair D. Toward a unified theory of caloric restriction and longevity regulation. Mech Ageing Dev 126: 987–1002, 2005.[CrossRef][Web of Science][Medline]
  38. Singhal A, Cole TJ, Fewtrell M, Lucas A. Breastmilk feeding and lipoprotein profile in adolescents born preterm: follow-up of a prospective randomised study. Lancet 363: 1571–1578, 2004.[CrossRef][Web of Science][Medline]
  39. Singhal A, Cole TJ, Fewtrell M, Deanfield J, Lucas A. Is slower early growth beneficial for long-term cardiovascular health? Circulation 109: 1108–1113, 2004.[Abstract/Free Full Text]
  40. Snoeck A, Remacle C, Reusens B, Hoett JJ. Effect of low protein diet during pregnancy on the fetal rat endocrine pancreas. Biol Neonate 57: 107–118, 1990.[Web of Science][Medline]
  41. Srivastava T. Nondiabetic consequences of obesity on kidney. Pediatr Nephrol 21: 463–470, 2006.[CrossRef][Web of Science][Medline]
  42. Tarry-Adkins JL, Ozanne SE, Norden A, Cherif C, Hales CN. Lower anti-oxidant capacity and elevated p53 and p21 may be a link between gender disparity in renal telomere shortening, albuminuria and longevity. Am J Physiol Renal Physiol 290: F509–F516, 2006.[Abstract/Free Full Text]
  43. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39: 44–84, 2007.[CrossRef][Web of Science][Medline]
  44. Van Remmen H, Ikeno K, Hamilton M, Pahlavani M, Wolf N, Thorpe SR, Alderson NL, Baynes JW, Epstein CJ, Huang TT, Nelson J, Strong R, Richardson A. Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging. Physiol Genomics 16: 29–37, 2003.[Abstract/Free Full Text]
  45. Vaziri ND, Dicus M, Ho ND, Boroujerdi-Rad L, Sindhu RK. Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency. Kidney Int 63: 179–185, 2003.[CrossRef][Web of Science][Medline]
  46. von Zglinicki T. Oxidative stress shortens telomeres. Trends Biochem Sci 27: 339–344, 2002.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. S. Martin-Gronert, J. L. Tarry-Adkins, R. L. Cripps, J.-H. Chen, and S. E. Ozanne
Maternal protein restriction leads to early life alterations in the expression of key molecules involved in the aging process in rat offspring
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R494 - R500.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. F. Schreuder
Combined nephroprotective effect and low nephron endowment as a consequence of postnatal growth restriction in the rat?
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R276 - R276.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. A. Joles, J. L. Tarry-Adkins, S. Snoeijs, D. M. van der Giezen, R. Goldschmeding, and S. E. Ozanne
Reply to: Schreuder
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R277 - R278.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/R1259    most recent
00231.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tarry-Adkins, J. L.
Right arrow Articles by Ozanne, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tarry-Adkins, J. L.
Right arrow Articles by Ozanne, S. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.