ACUTE UREMIA IN RATS: FEEDING SUCROSE OVERNIGHT IS PREFERABLE TO STARVATION

Abstract

Some experiments on uremia in rats may require an overnight fast in order to empty the gastrointestinal tract, or for other reasons. Previous investigation of normal rats showed that an overnight fast is not completely innocuous and that feeding pure sucrose is a valuable alternative. In the present study, anephric uremic rats were fasted or fed sucrose for 20 h. Sucrose feeding resulted in lower serum urea values and less loss of liver weight. Therefore, feeding sucrose overnight is preferable to starvation in uremic rats, as in normal rats.

• Uremia in rats
• Sucrose feeding
• GI tract
• Starvation

INTRODUCTION

Experiments on uremic rats often require overnight starvation. The purpose of the deprivation is usually the emptying of the gastrointestinal (GI) tract. An empty GI tract facilitates abdominal surgery and gastric intubation, reduces accidental penetration of stomach or cecum by intraperitoneal injections, provides basal conditions for blood chemistry, and avoids interference by food and digestive secretions with the absorption and metabolism of drugs or nutrients under study. However, overnight starvation may be particularly detrimental to rats because they are nocturnal feeders. Recently, we showed that feeding normal rats pure sucrose overnight empties the GI tract while avoiding or minimizing the loss of body and liver weights and depletion of liver glycogen and blood glucose that can be caused by brief starvation [[1]], [[2]]. In the present work, we have studied this problem in uremic rats in order to determine if feeding sugar overnight is preferable to overnight starvation when uremic rats are the experimental subjects.

MATERIALS AND METHODS

Male Lewis rats were raised in hanging shoe-box type plastic cages with hardwood litter. Tap water and Laboratory Rodent Diet 5001 (PMI Feeds, St. Louis, MO) were freely available. When the rats weighed 200–260 g, the right kidneys were removed. The anesthetic was ketamine and acepromazine injected intramuscularly. One week later, the left kidney was removed through the same dorsal midline incision with the aid of isoflurane anesthesia. The interval between nephrectomies allowed the right adrenal to recover from any injury that might have occurred during the first nephrectomy. The rats were housed in pairs.

Four or 28 h after the second nephrectomy, a metal grid was inserted into all the cages and all bedding was removed to reduce coprophagy and consumption of bedding material (pica) [[3]]. Each of the five experiments included three groups of four rats. One group was continued on its usual diet of 5001 pellets. The second group was deprived of all food. A third group was provided sucrose cubes (Domino Dots, Domino Sugar Corp., New York, NY) as sole nutrient. All cages had tap water freely available.

Twenty hours later, the rats were anesthetized with carbon dioxide and exsanguinated from the inferior vena cava. Livers and entire gastrointestinal tracts (including contents and esophagus) were weighed fresh. Serum was analyzed in a BMD Hitachi 736-30 auto-analyzer. Liver tissue, fixed in cold Bouin's fluid, was embedded in paraffin, sectioned, and stained by periodic acid-Schiff-hematoxylin. Stained material was presumed to be glycogen because pretreatment of the slides with diastase eliminated the staining.

Statistical Analysis

Each experiment was analyzed separately because they differed in starting time after onset of uremia. Two statistical analyses were utilized to test the global hypothesis of equality of all dietary treatments: a one-way analysis of variance (ANOVA) which assumed a normal distribution of data, and the non-parametric Kruskal-Wallis test. If ANOVA indicated that the treatments had significantly different effects, multiple comparisons among the groups were done by Dunnett's one-tailed procedure. This procedure protects the size of the overall type I error at 5%.

RESULTS

A preliminary experiment revealed that anephric rats could survive for 72 h if fasted or fed sucrose cubes, but only for 48 h if allowed their usual diet of 5001 pellets. To avoid mortality in the latter group, three experiments were started 4 h after the inception of uremia and concluded 20 h later, and two experiments were started 28 h after the inception of uremia and concluded 20 h later.

ANOVA and Kruskal-Wallis tests indicated overall significant differences in all three experiments studied 24 h after inception of uremia. All these rats had high values of urea in their serum, but it was lower in the starved and sucrose-fed rats than in the rats fed their usual diet of 5001 pellets and these differences were statistically significant (p<0.05) (Table 1). ANOVA and Kruskal-Wallis tests indicated overall significant differences in only one of the two experiments studied 48 h after inception of uremia. Now the serum urea values were all much higher. The urea values for the fasted rats were not significantly different from the values in rats fed the 5001 pellets, and the rats fed sucrose had significantly lower values only in one of the two experiments (Table 1).

Table 1. Overnight Starvation or Feeding Sucrose to Uremic Rats

Duration Uremia (h)	Diet Last 20 h	Serum Urea N (mg/dl)3^c	Liver Weight (% body)3^c
24	Rodent diet 5001	150±7	3.45±0.13
	No food1^a	131±54^d	3.27±0.04
	Sucrose cubes1^a	120±54^d	3.41±0.19
24	Rodent diet 5001	184±11	3.62±0.07
	No food1^a	155±34^d	3.25±0.04
	Sucrose cubes1^a	151±34^d	3.32±0.06
24	Rodent diet 5001	190±3	3.56±0.09
	No food1^a	155±54^d	3.51±0.06
	Sucrose cubes1^a	151±94^d	3.57±0.14
48	Rodent diet 5001	270±12	3.75±0.06
	No food2^b	257±11	3.80±0.06
	Sucrose cubes2^b	226±64^d	3.85±0.02
48	Rodent diet 5001	275±7	3.56±0.12
	No food2^b	276±8	3.46±0.10
	Sucrose cubes2^b	270±13	3.61±0.12

^aDietary changes instituted 4 h after second nephrectomy and lasted only 20 h.

^bDietary changes instituted 28 h after second nephrectomy and lasted only 20 h.

^cAverage values, four rats each group, ± standard deviations.

^dDunnett's test showed significant differences between starved or sucrose-fed groups and the control 5001-fed groups as indicated by the superscript (p<0.05).

The GI tracts (including contents) of the control rats fed their usual diet of 5001 pellets and studied 24 h after inception of uremia constituted an average of 9.86% of body weight. The average weight of the GI tracts of the fasted rats was 6.69% of body weight, and of the sucrose-fed rats was also 6.69% of body weight. This indicated that fasting and sucrose feeding were equally effective in emptying the GI tracts. As expected, the livers of sucrose fed rats were slightly larger than the livers of starved rats in all the experiments (Table 1). Histologically, the livers of sucrose fed rats had more periodic acid-Schiff positive material (eliminated by diastase and presumed to be glycogen) than the livers of starved rats.

DISCUSSION

Most experiments on anephric rats are done immediately after bilateral nephrectomy because of the short survival. When the planned experiment requires an empty GI tract, we propose feeding sucrose overnight rather than starving. The advantage of this procedure is indicated by the lower serum urea. A similar advantage accrued to normal rats and has been attributed to the reduced gluconeogenesis from amino acids and the absence of potassium and nitrogenous nutrients when sugar is provided as the only food [[1]], [[2]].

However, the advantages of sucrose feeding were variable in rats studied 48 h after onset of uremia. This may be related to the increasing metabolic disturbance as uremia progressed [[4]]. Also, uremic rats adapt to starvation differently than normal rats [[5]]. Even if the benefits were less, sucrose feeding may still be preferable to starvation, unless sugar is contraindicated by the experimental design.

In chronic experiments, sucrose has detrimental effects on uremic rats [[6]]. However, those effects are not pertinent to the brief overnight experiments described here. In normal rats, glucose and other sugars gave the same benefits as sucrose, provided they were consumed in adequate amounts [[2]]. We chose sucrose cubes because they are inexpensive and readily available, and can be used in conventional food hoppers.

We have described a procedure for producing anephric rats with longer survival (at least 6–8 days). Unfortunately, this method could not be applied to the present problem because it requires the emptying of the GI tract in advance of the second nephrectomy [[7]], [[8]].

ACKNOWLEDGMENT

We thank Morris Meisner and Shang Lin for the statistical analysis.