Acute Uremia Produced in Rats by Nephrotoxic Chemicals Is Alleviated by Protein Deficient Diet

Abstract

Rats injected with mercuric chloride develop an acute renal tubular necrosis with uremia, which is frequently lethal. Pretreatment for 3 or 7 days with a protein-free diet reduces the mortality, the clinical signs (tremor), and the severity of renal tubular necrosis, and ameliorates the uremic chemical findings in the serum. Similar results followed injection of a nephrotoxic amino acid, d-serine, after pretreatment with a protein-free diet. Indirect evidence suggests that induction of metallothionein may be involved, at least in the experiments with mercury. Acute uremia produced by nephrotoxic chemicals may be useful for further studies of the role of nutrition in uremia, while avoiding the surgical procedures and prolonged observations required for the “remnant kidney” models.

• Uremia
• Nephrotoxin
• Mercuric chloride
• d-Serine, Metallothionein
• Nutrition
• Protein deficiency

Introduction

Nutritional studies on experimental uremia in rats usually utilize subtotal nephrectomy in one or another of its variants.[[1]], [[2]] The uremia in subtotally nephrectomized rats develops gradually over a period of many weeks or months, which slows the progress of nutritional investigations. There are relatively few nutritional studies that make use of the acute uremia that develops immediately after administration of a nephrotoxic chemical.[[3]] In the present work, we show that nutrition can play an important role in the acute uremia produced in the rat by mercuric chloride or d-serine. We studied protein deficient diets including the pure carbohydrate diet that was very successful in prolonging the life span of bilaterally nephrectomized or ureter ligated rats.[[4]], [[5]]

Materials and Methods

Lewis rats obtained from Harlan–Sprague Dawley were bred in our laboratories. Male offspring were maintained on Laboratory Rodent Diet 5001 (PMI Nutrition International, Brentwood, MO) and tap water, both freely available. When they were 6–10 weeks old, some were fed a semi-synthetic protein-free diet (based on AIN 76A, Dyets, Inc., Bethlehem, PA) or sucrose cubes (Domino Dots, Domino Sugar Corp., New York, NY) as sole nutrient for one week, while others were continued on their usual diet. The cages of rats given special diets were provided with metal grids on the floor to reduce coprophagy. Mercuric chloride (HgCl₂) 1 mg/mL in distilled water, 0.5 or 1.0 mg/100 g body weight, was injected subcutaneously (SC) in the flank, once only. These doses are within the range used by previous investigators as reviewed by Tanner.[[3]] d-Serine, 53 mg/mL in distilled water, 3 mL/100 g body weight, was injected intraperitoneally on two successive days. The same diets were continued until necropsies were performed 4 or 2 days later. The rats were anesthetized with CO₂ and exsanguinated from the inferior vena cava. Serum was analyzed for urea and inorganic phosphorus in a Roche-Hitachi model 747-100 analyzer. Kidneys were fixed in Bouin's fluid, and a cross-sectional slice of the middle of each kidney, through the hilum, was embedded in paraffin, sectioned at 5 µ and stained with hematoxylin and eosin. The severity of tubular damage was scored as 5+ when almost all tubules in both inner and outer cortex were necrotic including the outermost (subcapsular) tubules, 4+ when the damage in outer cortex was less severe and affected few or none of the outermost (subcapsular) tubules, 3+ when necrosis was severe but restricted to the inner cortex, 2+ for partial damage in the inner cortex, and 1+ for minor changes in the pars recta of the proximal convoluted tubule, detected at high magnification (×400) (this score was rare in the present experiments). Slides were randomized and scored without knowledge of the experimental group. Scores were evaluated by the method of paired comparison.[[6]], [[7]]

Results

Four days after SC injection of HgCl₂ rats fed their usual, complete diet had very high levels of urea and inorganic phosphorus in their serum. Some of them had tremors. Histologically, their kidneys had very severe tubular necrosis (Table 1). Rats fed a protein-free diet had much less elevation of urea in their serum and none had tremors following HgCl₂ injection. Their kidneys had tubular necrosis, but it was much less than the necrosis noted in rats fed the control diet. However, the elevation of inorganic phosphorus in the serum was only minimally attenuated (Table 1).

Table 1. Effect of diet on nephrotoxicity of mercuric chloride

Diet	Dose of HgCl2	Urea N (mg/dL)	Inorganic P (mg/dL)	Histologic necrosis score	No. of rats
Complete	1 mg/100 g	488±28	37.8±5.4	4.8±0.3	12
Complete	0.5 mg/100 g	421±72	31.3±8.9	4.1±0.3	8
Complete	None	23±2	11.2±0.4	0.0	4
Protein-free	1 mg/100 g	233±33	28.2±3.1	3.0±0.0	8
Sucrose	1 mg/100 g	196±73	18.2±5.4	3.1±0.3	14
Sucrose	0.5 mg/100 g	141±53	14.6±2.8	2.9±0.3	11
Sucrose	None	9±2	13.7±1.0	0.0	4

Protein-free and sucrose diets started 7 days before HgCl₂ challenge and continued until necropsy 4 days later.

Serum chemistry results and scores are averages (± standard deviation). Histologic scores were evaluated by method of paired comparisons; coefficient of consistence was unity, indicating no inconsistencies.

Rats fed the sucrose diet also had much less elevation of urea, much less tubular necrosis, and none had tremors, but in addition, there was much less elevation of inorganic phosphorus (Table 1). As phosphates are significant uremic toxins, the remaining experiments were done with the pure sucrose diet rather than the diet deficient only in protein.

The sucrose-fed rats were started on their restricted diet one week before HgCl₂ was injected, and they lost considerable weight during that week. We considered the possibility that the sucrose diet reduced the severity of nephrotoxic injury simply because HgCl₂ was administered on a body weight basis, hence lighter animals received less HgCl₂. To explore this possibility, one of our experiments utilized two groups of sucrose-fed rats for each group of complete diet-fed rats. One of the sucrose-fed groups was selected 30–50 g heavier than the complete diet-fed rats so that they were approximately equal in average weight one week later when HgCl₂ was injected: 154 g and 149 g respectively. The second group of sucrose-fed rats were started on the restricted diet while they were in the same weight range as the complete diet-fed rats; consequently they were lighter than the complete diet rats when HgCl₂ was injected: average 108 g compared to average 149 g. When studied 4 days after HgCl₂ injection, the heavier and lighter sucrose-fed rats had similar uremic indicators (average serum urea nitrogen 218 and 212 mg/dL, inorganic phosphorus 18.4 and 20.0 mg/dL, histologic scores 3.0 and 3.5 respectively), always much less than the values in complete diet-fed rats (average urea nitrogen 481, inorganic phosphorus 37.7, scores 4.8) (each group four rats). These results have been confirmed in other experiments with the same or lower doses of HgCl₂. Therefore, the protective effect of sucrose diet was not merely the effect of lower body weight on HgCl₂ dosage.

For future use of sucrose feeding in uremia research, it was important to determine how long the rats had to be kept on the restricted diet. Therefore, groups of four rats were fed pure sucrose for 7 or 3 days before HgCl₂ treatment (0.5 mg/100 g). When studied 4 days after HgCl₂ both of these groups had far less elevation of urea and inorganic phosphorus and less tubular necrosis than the control groups fed complete diet (Table 2). However, 7 days on sucrose diet yielded better (lower) serum levels than 3 days on sucrose diet. Despite the differences in serum levels, these last two groups could not be differentiated histologically (Table 2).

Table 2. Seven days on sucrose diet is more effective than 3 days

Diet	Duration before challenge	Dose of HgCl2	Urea N (mg/dL)	Inorganic P (mg/dL)	Histologic necrosis score
Complete	—	0.5 mg/100 g	416 ± 51	33.7 ± 4.2	4.3 ± 0.3
Sucrose	3 days	0.5 mg/100 g	242 ± 50	24.7 ± 4.8	3.0 ± 0.0
Sucrose	7 days	0.5 mg/100 g	159 ± 39	15.5 ± 1.9	3.0 ± 0.0

Sucrose diets started 7 or 3 days before HgCl₂ (SC) and continued until necropsy 4 days later. N = 4. See footnotes for Table 1.

Nutritional imbalances are known to induce metallothionein, and metallothionein protects against mercury toxicity. In order to support the role of metallothionein in our results, it was important to show that similar results could be obtained by a different method of induction. For this purpose, ZnSO₄·7H₂O, 9 mg/100 g, was injected SC for five consecutive days before HgCl₂ challenge into groups of rats fed either sucrose or complete diet.[[8]] Comparison with control groups indicated that this classical inducer of metallothionein caused a slight reduction of HgCl₂ nephrotoxicity in rats fed the normal diet and a very impressive reduction in rats fed the sucrose diet (Table 3).

Table 3. Zinc sulfate reduces the nephrotoxicity of HgCl₂, especially in rats on sucrose diet

Diet	Pretreatment	Dose of HgCl2	Urea N (mg/dL)	Inorganic P (mg/dL)	Histologic necrosis score
Complete	None	1 mg/100 g	465±21	39.8±2.0	5.0±0.0
Complete	ZnSO4	1 mg/100 g	425±18	37.7±1.6	4.5±0.3
Sucrose	None	1 mg/100 g	174±26	16.3±1.1	3.0±0.0
Sucrose	ZnSO4	1 mg/100 g	37±7	10.9±1.0	2.0±0.0

Sucrose diet and five consecutive daily SC injections of zinc started 7 days before HgCl₂ challenge. Diets, but not zinc, continued for 4 days after HgCl₂ challenge, and then necropsies were performed. N = 4. See footnotes for Table 1.

All of the data described above were derived from rats studied 4 days after HgCl₂ injection. In a further study, necropsies were performed earlier, 1 or 2 days after HgCl₂ challenge (1 mg/100 g). At these early times also, the sucrose diet starting 7 days before HgCl₂ challenge reduced the elevations of serum urea and phosphorus and the histologic scores of tubular necrosis compared to rats fed the usual complete diet.

In an additional experiment, the parameter studied was survival after SC injection of 1.0 or 0.5 mg/100 g of HgCl₂ (data combined because there were no differences). Six out of 8 rats on the usual complete diet were dead by the fifth day after HgCl₂ challenge, and four of them had tremors on the fourth day. In contrast, eight rats on sucrose diet starting 7 days before HgCl₂ challenge had neither tremors nor mortality. They were necropsied 11 days after HgCl₂ challenge at which time the serum chemistry had returned to normal in all but one rat.

For comparison with HgCl₂, rats were given d-serine, a nephrotoxic d-amino acid, 160 mg/100 g intraperitoneally on two successive days. Necropsy one day after the second dose revealed average serum urea N 283 mg/dL in rats fed complete diet. This very high level was reduced to 111 mg/dL in rats fed pure sucrose diet starting 7 days before challenge. Similarly, average histologic scores of kidney damage were reduced from 4.0 to 3.5 by the sucrose diet (six rats each group).

Discussion

Diets with inadequate protein content usually increase susceptibility to toxic chemicals, but instances of decreased susceptibility are known.[[9]], [[10]] It is also well known that low protein diets retard the progression of chronic renal diseases with uremia[[11]], [[12]] and this has been attributed to reduced uremic toxins of dietary or endogenous origin.[[13]] Most of this literature concerns progression of glomerular damage, but uremic toxins damage renal tubules as well, and the mechanism has been partly elucidated.[[14]], [[15]]

The literature includes several studies on nutritional aspects of intoxication with organic mercury because of its importance as an industrial pollutant.[[16]] Low dietary protein tended to protect mice from methylmercury.[[17]] Organic mercury compounds like methylmercury are more neurotoxic than nephrotoxic, hence not pertinent to the present study. In contrast, there are very few studies on the effects of nutritional deficiencies on inorganic mercury and its nephrotoxicity.

The attenuation of HgCl₂-induced uremia by a protein-free diet can be attributed to the reduction of dietary uremic toxins and precursors, as is the case in uremia of end stage renal disease,[[11]], [[12]] and in experimental models of rats with remnant kidneys.[[18]], [[19]], [[20]] However, the remnant kidney model requires many weeks or months of observations. The HgCl₂ model presented herein is unique for its short duration, requiring only 3 or 7 days of preparatory dietary deprivation and only a few days of observation after HgCl₂ intoxication. For this reason, this model may be useful for further research on the contributions of specific dietary components and uremic toxins.

Nutritional deprivation of any type may be accompanied by nonspecific changes such as stress and induction of metallothionein.[[21]], [[22]] Metallothionein protects against heavy metals,[[8]] and is therefore significant for HgCl₂ intoxication, as indicated in Table 3. However, we had similar results with a nephrotoxic amino acid. We are not aware of any evidence that metallothionein protects against this nonmetallic nephrotoxin. A definitive conclusion will require assays of metallothionein and/or use of genetic models.

Feeding sucrose should not be equated with starvation. During starvation, body proteins are catabolized to provide amino acids for conversion to glucose (“gluconeogenesis”). Feeding sucrose avoids the need for gluconeogenesis and thereby conserves body protein and reduces production of nitrogen-containing toxins. Rats consume sucrose readily and will survive many weeks before a vitamin deficiency develops.