INTRODUCTION

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Acute renal failure is characterized by a deterioration of renal function over a period of hours to days, resulting in the failure of the kidney to excrete nitrogenous waste products and maintain fluid and electrolyte homeostasis. Acute renal failure is common in hospitalized patients, ranging from 1 to 15% (1). It is a common cause of morbidity, increased hospitalization costs (20 to 60% require dialysis), and increased mortality (rates range from 7 to 80%) (1, 2). Although there are many causes of acute renal failure, renal injury may result from decreased renal perfusion or an ischemic/toxic insult to the renal tubule (1, 3). Many clinical conditions can lead to renal ischemia as a result of either extrarenal or intrarenal compromise of renal blood flow (1). Rhabdomyolysis, common after trauma and burn injury, is one such example. Prevention and early correction of renal ischemia may prevent or blunt the degree of renal injury and improve outcome.

A variety of therapeutic approaches have been used in an attempt to prevent acute ischemic and nephrotoxic renal injury (4). Amino acids and protein act as vasodilators to renal vessels and also possess cytoprotective actions within the kidney (5). Thus, we hypothesized that enteral nutrition would protect the kidney from ischemic injury. This study was designed to investigate the effects of enteral nutrition on renal function and mortality during experimental rhabdomyolysis.

	METHODS

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The study was approved by our Animal Care and Use Committee and conforms to the standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23 [revised 1985], United States Department of Health and Human Services, Washington, DC).

Experiment 1. Survival. Male Sprague-Dawley rats weighing 350 to 400 g were anesthetized intraperitoneally with sodium pentobarbital (20 mg/kg) and intramuscularly with ketamine hydrochloride (60 mg/kg). They were then subjected to abdominal laparotomy and placement of duodenal feeding tubes. Animals were allowed free access to food (rat chow) and water and allowed to recover over night. The following morning they were randomized to receive an enteral infusion of tap water (n = 17) or a peptide-based enteral formula (n = 18) (Reabilan-HN; Nestlé Nutrition, Deerfield, IL) at 3 ml/h. The composition of the formula is listed in Table 1. This infusion rate meets greater than 100% of daily water needs. The amount of nutrition given to the diet group represents 75% of their normal nutrient intake (i.e., protein and calories). Animals received 96 Kcal/d and 4.2 g protein/d. Normal intake for these animals is approximately 130 Kcal and 5.5 to 6.0 g protein per day. Six hours after initiation of the enteral infusion, animals were injected into both hind limbs with 50% glycerol (10 ml/Kg intramuscularly). Glycerol-induced muscle necrosis is a welI-established model of acute renal failure in rats (11, 12). Survival was assessed daily for 3 d and blood samples were obtained from anesthetized survivors via cardiac puncture for measurement of blood urea nitrogen (BUN) and creatinine. Animals did not have access to additional food or water after initiation of enteral infusions. BUN and creatinine were measured using a Kodak Ektachem DT Analyzer (Eastman Kodak, Rochester, NY).

Experiment 2. Assessment of renal plasma flow (RPF) and glomerular filtration rate (GFR). Male Sprague-Dawley rats weighing 350-400 g received feeding tubes as in Experiment 1. The following day they were randomized to receive an enteral infusion of water (n = 7) or peptide-based enteral formula (n = 7) at a rate of 3 ml/h. Six hours after initiation of enteral feeding, all animals were injected with 50% glycerol. The following day, animals were anesthetized (pentobarbital plus ketamine) and an internal jugular catheter, a tail artery catheter, and a bladder catheter (13) were placed surgically. Renal function was assessed by measuring GFR using inulin clearance and RPF using para-aminohippurate (PAH) clearance (14). Animals were infused with [¹⁴C]PAH (0.136 µCi/ml) and [³H]inulin (0.193 µCi/ml) intravenously (7.4 ml/h) for a 1.5-h equilibration period (radioisotopes were purchased from Dupont/NEN, Boston, MA). Urine was then collected for two 1-h periods. Blood samples were collected at the midpoint of each urine collection. Urine and plasma samples were counted in a scintillation counter for presence of each radioisotope. [¹⁴C]PAH and [³H]inulin clearances were calculated and averaged for the two time periods.

Survival data are presented in the form of survival curves, and they were analyzed using a log rank test. BUN, creatinine, PAH clearances, and inulin clearances are presented as means ± SEM and were analyzed using two-tailed Mann-Whitney rank-sum tests; p < 0.05 was considered significant.

	RESULTS

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Experiment 1. Survival. Seventy-eight percent (14 of 18) of the animals receiving the enteral diet survived 3 d compared with 35% (six of 17) of the water-fed animals (p < 0.05) (Figure 1). In addition, BUN (47 ± 8 versus 137 ± 27 mg/dl) and creatinine (0.8 ± 0.1 versus 2.0 ± 0.3 mg/dl) concentrations were significantly lower in the diet group survivors compared with the water survivors.

View larger version (9K): [in this window] [in a new window]   Figure 1.   Survival curves of formuIa-fed (closed circles) and water-fed (closed squares) animals after glycerol injection. Curves are significantly (p < 0.05) different.

Experiment 2. Twenty-four hours after injury, RPF was significantly higher in the diet group than in the water group (4.83 ± 0.65 versus 2.37 ± 0.62 ml/min) (Figure 2). In addition, GFR was significantly higher in the diet group than in the water group (2.05 ± 0.27 versus 0.89 ± 0.22 ml/min) (Figure 3). Normal values in our laboratory are 5.63 ± 0.46 ml/min for RPF and 2.32 ± 0.22 ml/min for GFR.

View larger version (30K): [in this window] [in a new window]   Figure 2.   Renal plasma flow (ml/min) after glycerol injection (rhabdomyolysis) in animals receiving an enteral diet versus water. Asterisk indicates p < 0.05.

View larger version (26K): [in this window] [in a new window]   Figure 3.   GIomerular filtration rate (ml/min) after glycerol injection (rhabdomyolysis) in animals receiving an enteral diet versus water. Asterisk indicates p < 0.05.

	DISCUSSlON

The results of this study indicate that enteral administration of nutrients can improve survival and prevent renal damage after glycerol-induced muscle necrosis. Although previous studies of enteral feeding have found protection of the gut barrier (15) and liver (16) after shock, this is the first study to demonstrate protection of the kidney from ischemic injury with enteral nutrition.

Glycerol-induced renal injury is a well-established model of acute renal failure in the rat (11, 12). Intramuscular injection of glycerol reliably causes rhabdomyolysis and acute tubular necrosis. Renal blood flow, glomerular filtration rate, and urine output are reduced (11, 12). Morphologically, there is extensive renal tubular cell necrosis and plugging of tubules with casts and heme crystals. This model has many similarities to human acute tubular necrosis associated with muscle injury/trauma and massive hemolysis. Much of the renal damage is believed to result from ischemia.

Mannitol and saline administration (i.e., volume loading) prior to glycerol injection can improve survival and prevent renal failure (17, 18). These agents help maintain renal blood flow and limit ischemia. On the other hand, dehydration and volume depletion exacerbate the renal damage induced by rhabdomyolysis (19). Our animals received equal quantities of fluids prior to and during the study. However, it is possible that intravascular volume was higher in the diet group because of intake of nutrients and electrolytes. Thus, preservation of intravascular volume and maintenance of cardiac output and renal blood flow in the fed group may have prevented renal failure. Improved intravascular volume and hemodynamics could also blunt activation of the sympathetic nervous system, renin-angiotensin axis, and other vasoconstrictor systems. On the other hand, nutrient administration could augment vasodilator systems such as nitric oxide by providing the substrate arginine. The net effect would be maintenance of renal blood flow and decreased renal injury.

We have examined the early (4-h) effects of glycerol injection on blood pressure and urine output in two rats. After glycerol injection, there was a 40 mm Hg increase in mean arterial pressure over 2 h, with return to near normal levels by 4 h. Similarly, urine output increased over the first 3 h and returned to baseline by 4 h after glycerol injection. These results are compatible with activation of vasoconstrictor systems or inhibition of vasodilator systems. In fact, the finding of early hypertension has been previously described and may be caused by acute nitric oxide and plasma arginine depletion by circulating myoglobin (20), with resultant inhibition of endothelium-dependent vasodilation.

Amino acids and peptides derived from protein can improve renal blood flow. Protein (5) and amino acids (6, 10) possess vasodilatory effects on renal vessels and increase GFR (5, 6, 21). Vasodilatory influences of these agents may have prevented vasoconstriction within the kidney induced by rhabdomyolysis (22). In this regard, nitric oxide (NO) is an important renal vasodilator. Provision of arginine in the enteral formula may have improved renal NO synthesis and diminished renal ischemic injury. It is also possible that nutrients in the formula possess cytoprotective actions within the kidneys. Alanine has been reported to protect rabbit proximal tubules against anoxic injury (9), and glycine and glutathione are reported to protect renal tubules from hypoxia (8). It is also possible that amino acids enhanced renal recovery from acute tubular necrosis (7). The exact component of the formula that may be protective in this animal model requires further study.

Nutrient delivery may have prevented renal injury by protecting the gut barrier after muscle necrosis induced mesenteric hypoperfusion (which is common in patients with rhabdomyolysis). Myoglobin released from muscle tissue may scavenge nitric oxide in the bloodstream, further contributing to vasoconstriction of mesenteric vessels. Enteral feeding provides arginine for nitric oxide synthesis, dilates gut blood vessels, protects the gut barrier, and decreases bacterial/toxin translocation. These protective effects of enteral nutrition on the gut may diminish bacterial/toxin entry into the bloodstream and cytokine activation, both of which can cause renal injury (as occurs in sepsis).

This is the first animal study to address the effect of early feeding of a complete nutritional formula on renal function using a model of acute renal failure. Clinical studies of nutritional support in patients with acute renal failure are limited. There are no prospective randomized studies of enteral nutrition. However, in a small pilot study (23), increasing enteral protein intake was found to improve glomerular filtration in patients suffering from multitrauma.

Five clinical studies compared parenteral nutrition using amino acids and glucose to glucose alone in patients after the onset of renal failure. Three of the studies reported improved survival with parenteral nutrition using amino acids and glucose (24), whereas the remaining studies failed to find a significant improvement in outcome (27, 28). Interestingly, renal function improved in four of the studies (24).

The enteral formula used in this study contained both essential and nonessential amino acids. In the past, some have advocated the use of essential amino acids in patients with renal failure to minimize urea generation. We do not advocate this approach for numerous reasons. First, clinical studies comparing essential amino acid administration to general amino acids (essential and nonessential) have failed to demonstrate a benefit for essential amino acids on morbidity (including improvement in renal function) and mortality (29). Second, protein synthesis requires both essential and nonessential amino acids (28, 29). If these are limited, protein synthesis will be impaired. In addition, essential amino acid formulas do not support wound healing and immune function as well as general amino acid solutions. Third, a number of nonessential amino acids become essential during critical illness because of limitations in endogenous synthesis and mobilization from tissue stores. These include histidine, serine, arginine, taurine, cysteine, tyrosine, and glutamine (30). Finally, essential amino acid formulas are more expensive than formulas based upon intact proteins (containing essential and nonessential amino acids).

This study evaluated enteral nutrition prior to glycerol- induced injury. Prophylactic use of enteral nutrition may be useful in some clinical situations (i.e., surgery on trauma/burn patients with extensive tissue necrosis, cardiovascular surgery) where renal ischemia is common. It may also be useful in patients receiving drugs that cause renal ischemia such as amphotericin B, radiocontrast dye, and cyclosporin. Future studies are needed to determine if enteral nutrition after renal injury also improves renal function.

This report does not address whether there is any benefit of early nutrition in addition to the well-proved prophylactic value of early volume loading in preventing acute renal failure after rhabdomyolysis. Rhabdomyolysis and hemolysis after glycerol injection may result in severe hyperkalemia and hyperphosphatemia. Previously, we measured these electrolytes after glycerol injection. Values were within acceptable limits and should not have contributed to the high mortality in this study.

In summary, these results indicate that enteral feeding protects the kidney from ischemic injury and improves outcome after muscle injury. Outcome may have been improved because of systemic or renal protective effects of enteral feeding or both in combination. Enteral nutrition may have improved cardiac output by maintaining better intravascular volume, thus preventing or ameliorating systemic hypoperfusion. Secondary renal hypoperfusion would be minimized not only by improving cardiac output but also by blunting activation of neurohumoral responses (i.e., renin-angiotensin, catecholamine secretion) and possibly provision of arginine as a substrate for synthesis of nitric oxide (vasodilation). Enteral feeding may also provide agents with cytoprotective effects (i.e., amino acids, antioxidants) and may have protected the gut barrier, minimizing bacterial and toxin entry. Further studies are required to determine if enteral feeding has protective effects in humans at risk for acute renal failure.