Background

Female obese (fa/fa) Zucker rats are maximally hyperphagic from the beginning of access to solid food until 20 weeks of age and die primarily from renal failure. We documented that urinary albumin excretion (UAE) rises early in obese rats during this time of greatest hyperphagia. This study was conducted to examine if this early surge of hyperphagia is critical to the initiation of glomerular damage.

Methods

Three groups of six-week-old rats were used: (a) obese females fed ad libitum (AL-obese), (b) obese females pair fed to lean controls until 21 weeks and then allowed to eat ad libitum until 57 weeks (PF.AL-obese), (c) lean (Fa/Fa) Zucker rats fed ad libitum (AL-lean). Cohorts of AL-obese and PF.AL-obese rats were allowed to continue to death or 57 weeks of age, and the rest were terminated at 21 weeks for renal histology.

Results

At 21 weeks, neither PF.AL-obese nor AL-lean rats had elevated UAE or glomerular histopathology. In contrast, glomerular injury was severe in AL-obese rats. UAE increased by 10 and 29 weeks in AL- and PF.AL-obese rats, respectively. Plasma triglycerides increased prior to UAE in both PF.AL- and AL-obese rats.

Conclusions

In obese rats fed ad libitum, hyperphagia is followed within a few weeks by hypertriglyceridemia and then by glomerular injury regardless of when ad libitum feeding is initiated. These events do not occur in lean rats or in obese rats pair fed to lean rats. Protective effects of pair feeding did not extend into the period of ad libitum feeding for PF.AL-obese rats. Hyperphagia quickly initiates glomerular injury in obese female Zucker rats.

Methods

Experimental animals

Thirty obese (fa/fa) and eight lean (Fa/Fa) female Zucker rats were started on the study at six weeks of age. Rats were housed individually in suspended wire mesh cages and had free access to water. The light cycle was on at 7 a.m. and off at 7 p.m. Three groups of rats were studied. One group of obese females was allowed to eat ad libitum (AL-obese) from 6 to 57 weeks or until death (N = 15). A second group of obese females was pair fed to lean rats from 6 to 21 weeks of age and was then allowed to eat ad libitum to 57 weeks or until death (PF.AL-obese, N = 15). The third group was lean female Zucker rats fed ad libitum (AL-lean) from 6 to 21 weeks (N = 8). Ten AL-obese, 10 PF.AL-obese, and all AL-leans were killed at 21 weeks of age for renal histology. The inclusion of these latter two groups allowed us to establish the effect of food restriction (pair feeding) on prevention of renal injury at the time of initiation of ad libitum feeding. In the PF.AL-obese group, one rat died at 45 weeks. Three rats died in the AL-obese group at the 34th, 50th, and 57th week. Data taken in the two weeks prior to death were omitted for these rats.

Diet

All groups were fed a soy protein-based diet (20% wt/wt), which was shown to minimize diet-induced nephropathy and was previously used in our work^7,13,14. The diet of the PF.AL-obese rats during the period of restriction was vitamin enriched relative to that of ad libitum-fed animals, with the weight of the vitamin mix being increased from 2.0 to 3.33% of the diet. The mineral content was unchanged. Food cups were changed between 800 and 1000 hours. Body weights for obese rats were measured twice a week throughout the study. Food cups for obese rats were removed, weighed, and refilled twice a week for all ad libitum-fed rats and daily during the period of restriction for PF.AL-obese. Food intake was determined from the difference in weight of food cups minus spillage. Pair feeding was achieved daily by feeding the PF.AL-obese rats daily one seventh of the weekly average amount eaten by lean (Fa/Fa) female Zucker rats of the same age. Food intake data for lean rats (g/day) were provided from the group of 45 lean female Zucker rats eating the same diet in a previous study so that these groups could be run in parallel⁷.

Plasma triglycerides and cholesterol

One half of a milliliter of blood was collected in heparinized capillary tubes from each rat by tail bleed after an overnight fast at 10, 15, and 20 weeks of age for 10 AL-obese and 10 PF.AL-obese rats, at 20 weeks for AL-lean rats, and at 10, 20, 25, 29, 33, 37, 41, 47, 51, and 55 weeks of age for 5 PF.AL-obese and 5 AL-obese rats. Plasma was removed from the centrifuged blood and stored at -85°C until assayed. Plasma triglyceride and cholesterol levels were determined using enzymatic colorimetric kits from Sigma Chemical Co. (St. Louis, MO, USA).

Urinary and plasma albumin

One day before every blood collection, animals were put in stainless steel metabolic cages. Water was available freely, as was food, moistened to minimize spillage, except for the PF.AL-obese group, which was given only its prescribed amount of food. Urine was collected for 24 hours in flasks containing three drops of a 10% sodium azide solution to inhibit bacterial growth. Urine volumes were measured, and albumin concentration was determined by electroimmunodiffusion using rabbit anti-rat albumin as previously described¹⁵ to calculate the 24-hour UAE. At 29 weeks of age, plasma samples were ultracentrifuged to remove lipids, and the albumin concentration in the lipid-free fraction was determined. Albumin clearance was calculated from plasma albumin and UAE data.

Renal histology

Histology was performed on kidneys from 10 AL-obese, 9 PF.AL-obese, and 8 AL-lean rats killed at 21 weeks of age. Following exsanguination under anesthesia, rats were infused via the abdominal aorta with phosphate-buffered saline (0.1 M PBS, pH 7.4) containing 6% sucrose and 500 U heparin/liter for a sufficient time to blanch the kidneys. A polar region was removed for histochemistry and was flash frozen in liquid nitrogen. A 0.05% glutaraldehyde/4% paraformaldehyde in PBS fixative was then perfused for one minute or until the kidneys became rigid. The fixative was removed by reperfusion with the PBS/sucrose/heparin solution. Fixed tissue was then processed in glycol methacrylate for light microscopy as described¹⁶. Sections were stained by the periodic acid-Schiff technique. Glomeruli were scored by the method of Raij, Azar, and Keane, as previously reported¹⁷. Thirty to 40 glomeruli were scored from each kidney for three variables: focal and segmental glomerulosclerosis (FGS), mesangial matrix expansion (MME), and visceral epithelial cell adhesion formation to Bowman's capsule (adhesions). Each glomerulus was scored semiquantitatively on a scale of 0 to 4 for each variable, depending on the percentage of the glomerulus involved. A final score was obtained by multiplying the degree of injury by the percentage of glomeruli with that degree and adding the scores. The maximum score obtainable for each rat was thus 400. FGS was scored positive when MME, adhesion formation, and capillary obliteration were present in the same segment of the glomerulus. Histochemical detection of neutral lipid with Oil Red O was performed on frozen tissue slices using the methods previously described¹⁸. The method of Raij et al was used to score Oil Red O staining¹⁷.

Statistical analysis

Data were analyzed using Sigma Stat 2.0, a commercial statistics program (Jandel, San Rafael CA, USA). Data were first analyzed for normality. Plasma triglyceride and cholesterol, renal albumin clearance, and UAE required log transformation to achieve normality. Data were then analyzed for differences using the unpaired t-test when only ad libitum-fed and pair fed obese rats were compared. When AL-lean and the two obese groups were compared, Kruskal–Wallis one-way analysis of variance on ranks was used because variances were unequal. This was followed by post hoc analysis with Dunn's test for multiple comparisons when differences were detected. Differences were considered statistically significant at P 0.05.

RESULTS

Body weight and food intake

Average weights of ad libitum and pair-fed obese rats were the same at the start of the experiment, but by 10 weeks, pair-fed obese rats were 38% lighter Figure 1. At 31 weeks, the weights of PF.AL- and AL-obese groups did not differ. Differences in food intake reached a maximum at seven weeks, when pair-fed obese rats ate 45% less than food per day than AL-obese. By 21 weeks, the restriction was only 26%. The average restriction for the pair-fed obese rats over the period from 5 to 21 weeks was 31%. When unlimited food became available to PF.AL-obese rats at 21 weeks, there was no overcompensation in their food intake Figure 2. Indeed, food intake in PF.AL-obese rats did not even increase until five weeks later, at which time it rose only to a level equal to that of AL-obese rats. After 47 weeks, the average food intake dropped for the AL-obese group because of morbidity.

Figure 1.

Average body weights for ad libitum-fed obese (; AL-obese) and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (; PF.AL-obese) female Zucker rats. Data are mean SE.

Full figure and legend (14K)

Figure 2.

Average daily food intake for ad libitum-fed obese (; AL-obese) and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (; PF.AL-obese) female Zucker rats. Data are mean SE.

Full figure and legend (16K)

Urinary albumin excretion

Urinary albumin excretion in AL-obese rats exceeded the normal range of less than 1 mg/day¹⁹ at the earliest measurement, 10 weeks of age. In contrast, UAE remained normal in PF.AL-obese rats as long as food was restricted Figure 3. No AL-lean rats exceeded 1 mg/day UAE, and at 20 weeks, they were indistinguishable from the pair-fed obese rats Figure 4. The three highest and earliest individual maximums were from the AL-obese group.

Figure 3.

Average 24-hour urinary albumin excretion (UAE) for ad libitum-fed obese (; AL-obese) and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (; PF.AL-obese) female Zucker rats. Data are mean SE. *P < 0.05 for AL vs. PF-/PF.AL-obese.

Full figure and legend (16K)

Figure 4.

Range, and 25th, 50th, and 75th percentiles of 24-hour urinary albumin excretion in ad libitum-fed obese (AL-obese, N = 15) and obese pair-fed compared to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (PF.AL-obese, N = 15), ad libitum-fed lean (AL-lean, N = 7) female Zucker rats. Groups with different letters differ significantly (P < 0.05). Data are mean SE.

Full figure and legend (14K)

Plasma triglycerides and cholesterol

At 20 weeks, plasma triglycerides in the pair-fed obese rats did not differ from those of the AL-lean rats, whereas that in AL-obese rats was much higher Figure 5. Over the course of the study, plasma triglycerides became extremely high in AL-obese rats: four of five exceeded 5000 mg/dl, with one reaching 11,200 mg/dl. Pair feeding prevented this rise in triglycerides, whereas triglycerides in PF.AL-obese rats rose following the termination of food restriction at 21 weeks Figure 6. Differences in plasma cholesterol between AL-obese and PF.AL-obese rats became significant only after 25 weeks. Although the average cholesterol rose in AL-obese rats to a maximum of 640 185 mg/dl, that in PF.AL-obese remained stable throughout, near the overall average of 168 12 mg/dl Figure 7. Looking at all time points up to 55 weeks of age, four AL-obese rats exceeded 400 mg/dl, one of which exceeded 1200 mg/dl. In contrast, only one PF.AL-obese rat exceeded 400 mg/dl. At 20 weeks, cholesterol in pair-fed obese rats was lower than in AL-obese but higher than in AL-lean Figure 8. The four highest maximum individual triglyceride and cholesterol levels were in the AL-obese group.

Figure 5.

Average fasting plasma triglycerides for ad libitum-fed obese (; AL-obese) and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (; PF.AL-obese) female Zucker rats. Data are mean SE. *P < 0.05 for AL vs. PF-/PF.AL-obese.

Full figure and legend (15K)

Figure 6.

Range, and 25th, 50th, and 75th percentiles of fasting plasma triglycerides in ad libitum-fed obese (AL-obese, N = 15) and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (PF.AL-obese, N = 15), ad libitum-fed lean (AL-lean, N = 7) female Zucker rats. Groups with different letters differ significantly (P < 0.05). Data are mean SE.

Full figure and legend (12K)

Figure 7.

Average fasting cholesterol for ad libitum-fed obese (; AL-obese), and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (; PF.AL-obese) female Zucker rats. Data are mean SE. *P < 0.05 for AL vs. PF-/PF.AL-obese.

Full figure and legend (13K)

Figure 8.

Range, and 25th, 50th, and 75th percentiles of fasting plasma cholesterol in ad libitum-fed obese (AL-obese, N = 15) and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (PF.AL-obese, N = 15), ad libitum-fed lean (AL-lean, N = 7) female Zucker rats. Groups with different letters differ significantly (P < 0.05). Data are mean SE.

Full figure and legend (12K)

Plasma albumin

At 29 weeks, AL-obese plasma albumin was significantly lower than that in PF.AL-obese rats (22.2 3.5 vs. 37.2 1.8 mg/ml, P < 0.01; Figure 9). Renal albumin clearance was significantly higher in AL-obese rats Figure 9.

Figure 9.

Individual values for plasma albumin and renal albumin clearance values at 29 weeks of age in ad libitum-fed obese (; AL-obese), and obese pair-fed to lean up to 21 weeks of age and then ad libitum-fed to 57 weeks (; PF.AL-obese) female Zucker rats.

Full figure and legend (12K)

Insulin

At all time points measured, all obese rats were hyperinsulinemic. At 15 and 25 weeks of age, AL-obese and PF.AL-obese insulin levels were not different Table 1. At 20 weeks, PF.AL-obese insulin values were actually higher than those in AL-obese.

Table 1 - Fasting plasma insulin (pmol/liter) in female Zucker rats.

Full table

Histology

At 21 weeks, AL-obese rats showed strong evidence of glomerular pathology, whereas PF.AL-obese and AL-lean showed none. Al-obese rats had marked MME, and this, as well as FGS, and adhesions of the visceral epithelium to Bowman's capsule were higher than in PF.AL-obese or AL-lean rats (Table 2 and Figure 10). In addition, Oil red O staining showed very strong glomerular deposition of neutral lipids in AL-obese but not PF.AL-obese and AL-lean rats (Table 2 and Figure 10). Interstitial fibrosis was present in AL-obese rats only. In both AL-obese and PF.AL-obese rats, but not AL-lean rats, thickening of the arterial media and sporadic intimal thickening was observed.

Figure 10.

Representative glomeruli from (A and B) in ad libitum-fed obese (AL-obese; C) pair-fed obese (PF-obese), (D) ad libitum–fed lean (AL-lean) Zucker rats. Oil Red O staining on representative glomeruli from (E) pair-fed obese (PF-obese) and (F) ad libitum-fed obese (AL-obese) female Zucker rats at 21 weeks of age.

Full figure and legend (543K)

Table 2 - Renal histopathology and histochemistry in female Zucker rats at 21 weeks of age.

Full table

DISCUSSION

The most remarkable finding in this study is that pair feeding completely prevented glomerular damage. We knew from previous work that pair feeding decreased deaths attributed to end-stage renal disease in the obese Zucker rats⁷. Other studies with the female obese Zucker rats showed that food restriction from 11 to 40 or 50 weeks of age decreased the severity of glomerulosclerosis²⁰, and intermittent carbohydrate restriction from 6 to 30 or 33 weeks of age was found to decrease UAE but not MME²¹. Our data show that pair feeding to 21 weeks was more effective than either. Histological and Oil Red O scores were zero or nearly zero, and UAE was in the normal range for all PF.AL-obese rats at 21 weeks of age in this study. Thus, glomeruli from PF.AL-obese rats at this age were comparable to those of AL-lean rats.

In this study, the dramatic effects of pair feeding were illustrated at two time points in particular. First, the AL-obese group had a median UAE of 15 mg/24 hr at 10 weeks, whereas the pair-fed rats all had a normal UAE of less than 1. Thus, only four weeks of hyperphagia led to glomerular damage. Second, although the PF.AL-obese group was allowed to eat ad libitum after 21 weeks, the average food intake did not increase until 27 weeks; the median UAE at 25 and 29 weeks was 1.8 and 93 mg/24 hr. Clearly, hyperphagia led to glomerular injury after only a few weeks, even when it was delayed until the rats were half a year old. Thus, it appears that the early phase of hyperphagia is not critical for glomerular damage to occur.

We also suspected that reductions in plasma lipids were responsible for the renal-protective effects of pair feeding. In agreement, plasma triglycerides became elevated prior to or at the same time as UAE became abnormal at both time points mentioned earlier in this article, and the correlation between plasma triglyceride levels at 10 weeks and MME scores at 21 weeks approached significance for the AL-obese rats. Plasma cholesterol levels followed a different pattern. In PF.AL-obese rats, the average plasma cholesterol remained stable throughout the study. In AL-obese rats, however, levels became very high, peaking at 29 weeks, a time when UAE was at a maximum and plasma albumin was reduced. This sequence of events suggests that the nephrotic syndrome was the cause, rather than the consequence, of hypercholesterolemia²². It is likely that triglycerides increased first because of hyperphagia, whereas the more severe increase later in AL-obese rats was, like hypercholesterolemia, a result of the nephrotic syndrome.

High-protein diets have been shown to accelerate glomerulosclerosis in models of reduced renal mass, and they have long been known to exacerbate existing nephropathy²³. We do not believe that the effects of pair feeding are due to reduced protein intake, however, because both groups had the same moderate (20%) protein content in their diets, and the source was soy, not the casein typically used in high-protein studies. Our soy-based diet was chosen specifically because it reduced nephropathy and extended the lifespan in rats relative to casein-based diets¹³.

Another effect of food restriction can be normalization of hyperinsulinemia²⁴. The vascular endothelium^25,26, and particularly rat mesangial cells, may have a pathological response to hyperinsulinemia. The insulin-enhancing drug rosiglitazone was found to protect against nephropathy in obese male Zucker rats, although plasma lipids were reduced as well as insulin, and the mechanism of action was unclear²⁸. Here, fasting plasma insulin was not lower in pair-fed groups at any point. Thus, we found no evidence that insulin could have contributed to endothelial dysfunction in this model.

We have previously reported that estrogen exacerbates hypertriglyceridemia and accelerates renal damage, whereas ovariectomy attenuates both¹⁴. Ovariectomized rats ate the most, and estrogen-treated rats ate the least, apparently disconnecting hyperphagia from renal injury. Food restriction can delay puberty in the female rat by suppressing leutinizing hormone pulse amplitude²⁹. This would, in turn, decrease estrogen levels. However, this effect has not been demonstrated in the female obese Zucker rat, which has delayed puberty and is infertile³⁰. A more likely explanation is that both treatments that reduced glomerular damage also reduced plasma triglycerides. Ovariectomy did not lower the triglyceride level quite to that of leans nor did it quite keep UAE in the normal range at 21 weeks, but pair-feeding did. Although proteinuria in AL-obese rats at 21 weeks of age was already too severe to distinguish between initiation and progression, the massive lipid deposition is consistent with lipid/lipoprotein-induced injury.

The obese Zucker rat is a valuable model for the study of kidney disease because glomerulosclerosis occurs spontaneously and is not present in lean littermates. However, the number of physiological abnormalities caused by the fatty mutation and the confounding interdependencies between them have impeded the isolation and identification of the factor(s) initiating glomerular damage. We believe that the pair-feeding design, which we demonstrate to completely prevent renal damage in female obese Zucker rats up to 21 weeks of age, significantly narrows the field of possibilities. Pair feeding of obese Zucker rats corrects only some of the pathology. For example, the percentage of body fat, insulin, and adipose lipoprotein lipase activity in the obese Zucker rats is unchanged by pair feeding³¹. Thus, the physiological disparity between experimental and control groups introduced by pair feeding is minimized and likely smaller than that caused by surgical or pharmacological interventions such as nephrectomy, angiotensin-converting enzyme inhibition, streptozotocin-induced diabetes, puromycin aminonucleoside- or adriamycin-induced nephrotic syndrome, and high-cholesterol diets.

In humans with diabetes, nephrotic range proteinuria, chronic renal failure, and higher cardiovascular mortality are predicted by the presence of microalbuminuria³². Obesity, even without diabetes or hypertension, is associated with an increased incidence of microalbuminuria^33,34. Because the incidence of obesity in the population is high and increasing, it is important to understand the mechanism by which microalbuminuria is initiated so that excess mortality and morbidity in this expanding patient population can be minimized. Furthermore, normotensive, nondiabetic humans with microalbuminuria have higher triglycerides³⁵, and hyperlipidemia is an independent risk factor for the development of albuminuria in patients with diabetes^36,37. Thus, the close relationship between hyperlipidemia and glomerular damage found here appears to hold for humans as well. Also, as with the rats in this study, food restriction in patients with NIDDM can reduce proteinuria³⁸. For these reasons, we feel that the obese Zucker rat is particularly well suited for the investigation of the mechanism of initiation and progression of albuminuria in obesity or "syndrome X," particularly with respect to the role of hyperlipidemia, and that such investigations are of significant relevance to public health.

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Acknowledgments

This work was supported by the research service of the Department of Veterans Affairs and National Institutes of Health grants (NIA/DK 09945, T32 DK7355, DK 35747), the Nora Eccles Treadwell Foundation, and a Nestle Fellowship to M. Gades.