Chronic renal failure in the rat

BRI EF COM M U N I CAT I ON S Chronic Renal Failure in the Rat A Surgical PHILIP D. Model for Long-Term Acorn, MALCOLM Key Words: Renal failure; R. OGBORN, Ischemic; Toxicological Studies AND JOHN F. S. CROCKER Toxicological studies I N T RODU CT I ON The rat has proved a useful model in many renal physiological and pathophysiological studies. The majority of these involve the assessment of acute or chronic changes on a previously healthy animal. It is often difficult to extrapolate these findings to the situation of chronic renal failure. Renal function may play a major role in the excretion of intrinsic or extrinsic chemicals. Normal renal physiologic responses may compensate or exacerbate changes in fluid and electrolyte status, circulating volume, or acid-base status. Such a difficulty has arisen in this laboratory in the study of a possible relationship between chemicals known to leach from dialysis tubing and the acquired cystic disease seen in the kidneys of dialysis patients (Cracker et al., 1986). Diethyl hexyl phthalate, a marker for the chemicals, is rapidly excreted by the normal kidney, but accumulates in inverse proportion to renal function (Chen et al., 1978). This report describes a surgical technique for the induction of a predictable decline in renal function in the laboratory rat over a period of 6 months or longer, enabling long-term studies in physiology, pharmacology, and metabolism in evolving uremia. M AT ERI ALS AN D M ET H ODS Immature, outbred Sprague Dawley rats (150-200 g) were purchased from Charles River Canada (Quebec, Canada). Animals were housed in the Animal Care Centre of The lzaak Walton Killam Hospital for Children at 22°C on a 12-hr light/l2-hr dark cycle, under conditions in accordance with the Canadian Council on Animal Care. The surgical procedure was approved by the attending veterinary surgeon, Dr. J. Love, and the Animal Care Committee of Dalhousie University. The rats had free access to water and Rodent Laboratory Chow No. 5001 (Ralston Purina, St. Louis, MO), containing approximately 25% crude protein. From the Children, Addre ss enue, Department Halifax, re print Halifax, Received Nova Canada. to : Dr. re q ue sts Nova March of Pediatrics, Scotia, Scotia, Dalhousie University and the lzaak Walton Killam Hospital for zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH John F. S. Cracker, I.W.K. Hospital for Children, 5850 University Av- B3J 3G9 Canada. 12, 1986; revised and accepted December 2, 1986. 81 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Pha rm a c o lo g ic a l Me tho ds 18, 81- 88 (1987) Journalo f 0 1967 Else vie r Sc ie nc e Pub lishing C o ., Inc ., 52 Va nde rb ilt Ave nue , Ne w Yo rk, NY 10017 82 P. D. Acott et al. Aorta Right Kidney (Vascular Pedicle completely\sectioned) 1I 1 Area of lschemia Ligature applied to inferior division of artery to lower pole Technique of renal ischemia reduction. zyxwvutsrqponmlkjih FIGURE 1. SURG IC AL PRO C EDURE Anesthesia was induced by intramuscular kg body weight, and droperidol/fentanyl duced anesthesia adequate procedure. small A 6-cm bowel of ketamine (Ketalar), 0.25 ml/kg body weight. for 30 min, which was the time necessary midline and cecum injection (Inovar), ventral were incision lifted was made and placed for the surgical in the abdominal on saline-soaked 40 mg/ This pro- gauze wall. The sponges. The exposed right kidney was dissected from the retroperitoneal area and the vascular and ureteric pedicles were ligated with 2-O silk sutures and transected, and the kidney was removed. The renal artery of the left kidney hilum to expose the three main segmental not dissected out of the retroperitoneum. was then area % of the tory, temporarily of ischemia kidney, abdomen, 4-O dexon returned to establish demarcated a permanent the temporary area of ischemia ligated became ligature was produced. the volume within ligature would was dissected into the renal arteries (Figure I). The kidney was The anterior caudal branch of the artery IO-IS was placed. be moved The viscera along were of renal tissue supplied. s. If this approximated If the result was not satisfac- the artery then The %I to until a satisfactory carefully replaced in the and peritoneum and linea alba were closed en masse with a continuous suture. The skin was closed with stainless-steel clips, and the animal was to its original the conclusion PO STO PERATIVE cage for recovery. Rats were usually active within 30 min of of the procedure. A SSESSMENT Body weight was measured at regular intervals over a 12-month period. Weight loss was considered a sign of symptomatic uremia. Animals that became clinically ill were killed, and renal tissue was obtained for histological analysis. Blood for serum creatinine was collected by retroorbital puncture under anesthesia at 0, 6, 8, and 12 months after the procedure. In association with this, 24-hr urines were collected in Nalgene metabolic cages for measurement of creatinine, specific gravity. Creatinine clearance was determined from the formula protein, and UV/P without Chronic Renal Failure in the Rat 0 0 -, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE ( ) 12 TIME POST SURGERY 6 months FIGURE 2. Creatinine and creatinine clearance in surgically treated and control rats over a l-year period. for body mass. Creatinine was measured by the Jaffe reaction (Lustgarten and Wenk, 1972) in a routine service laboratory. Urine specific gravity was determined with a refractometer (Osmette, Japan), and protein was determined by the method of Bradford (1976). At 12 months postsurgery, the remaining rats were killed, and kidney tissue was cultured for bacterial and fungal growth and processed for histology. adjustment STATISTICAL 83 zyxwvu METHODS Data were compared between surgery and control groups using one-way analysis of variance (Hewlett Packard Statistics Package 1983). The onset of terminal uremia 0.04 1 RECIPROCAL OF CREATININE AS A PREDICTOR OF TERMINAL UREMIA . . . . ‘F ;\ .,Y = 0.0237-0.0015X '. 0.00-l , 0 6 TIME POST SURGERY 1 I 8 ( 12 months ) FIGURE 3. Previous experience has found that most rats die when serum creatinine reaches 500 ~mollliter (reciprocal = 0.002). The regression line intercepts this Y value at 14 months. 84 P. D. Acott et al. “y-Y---l zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ TIME POST SURGERY FIGURE 4. Measures of urinary concentrating 6 zyxwvutsrqponmlkjihgfedcbaZYXWVU a 12 zyxwvutsrqponmlkjih ( months ) ability in surgically treated and control rats. was predicted by the study of the inverse of the serum creatinine according to the method of Mitch et al. (1976). This method uses least-squares regression of the inverse of observed creatinine values versus time to predict the point in time at which creatinine will reach the range consistent with terminal uremia. A serum creatinine of 500 mm/liter was taken as the terminal point for the purpose of this study. The regression analysis was performed on the general statistics package, Minitab (Pennsylvania State University) on the Cyber computer facility of Dalhousie University. 0 6 8 12 TIME POST SURGERY ( months ) FIGURE rats. 5. Protein concentration of 24-hr urine collections in surgically treated or control 85 FIGURE 6. Renal tissue from a nonuremic animal 2 months after surgery (x50). Clomerular and tubular structures demonstrate marked hypertrophy. The section is from the nonischemic area. FIGI JRE 7. Renal tissue from an animal with evolving uremia 8.5 months postsurgery :x50). This tissue from a nonischemic area shows thyroidization with glomerulosclerosis and nnarked inte rstitial infiltrate. Chronic Renal Failure in the Rat 87 RESULTS In the initial series of experiments on DEHP absorption in uremia, 98 rats underwent surgery. Of these, 10 rats (10.2%) died for causes attributable to the procedure. Six died after administration orrhage. Three other of anesthesia, rats died and one died from intraoperative in the first 7 days after surgery hem- of uncertain cause. Two rats were found dead at approximately 7 months; postsurgery and preterminal studies are not available. The effect of surgery alone was evaluated in seven rats who did not receive potentially nephrotoxic chemicals. Four to five rats underwent study at each interval. Baseline values were determined in these rats and in zyxwvutsrq other 11 rats who did not undergo at the same time. who underwent nonoperated aspects Four of these surgery showed rats. The surgery of renal function. marized in Figure surgery and were latter rats were patterns 2A, showed of weight produced Serum bought at 12 months. gain similar significant creatinine from the same supplier also studied progressive in the surgically that surgically treated The rats to those of healthy changes treated in other rats, as sum- rats had significantly higher creatinines (p < .OOl) at 12 months than did control rats. Converse changes were seen with creatinine clearance (Figure 2B) (p < .OOl). When the inverse of the serum creatinine was plotted against time (Figure 3), a significant linear relationship was found (p < .OOl), and terminal Surgically treated rats showed uremia was predicted at 14-15 months postsurgery. highly significant increases in urine voume (Figure 4B, gravity decreased p < and urine specific .OOl), providing centration rats (Figure 5) (p < .OOl). The proteinuria in urine specific gravity than that which All bacterial onstrated medulla significantly (Figure4A, p 0.025) < further evidence of decreased concentrating ability. Urine protein conin a 24-hr collection was significantly increased in the surgically treated cultures extensive were negative. probably masked an even greater was actually observed. Microscopically, but focal areas of tubular of the infarcted segment (Figures necrosis the kidney decline remnants dem- of the inner cortex and outer 6 and 7). There was thyroidization inflammatory infiltrate adjacent to the areas of ischemic necrosis. was well demarcated from the nonischemic renal tissue. The infarcted and area DISCUSSION The course sistent described consistent with data demonstrate with survival that this model the progressive to an appreciable Stein et al. (1984) in a shorter used resection diathermy term to ablate course portion of uremia of chronic of the normal study have used a similar the lower pole produces renal failure, of the a clinical yet is con- life span of the rat. approach, remaining but they kidney and subsequently demonstrated considerably more rapid deterioration and greater mortality than this model. This may be a result of lack of control of the amount of tissue damage by diathermy. In this model, very little anatomical supply allowed accurate control of tissue destruction. Other variation described in vascular studies in- volve surgical removal of 50, 80 and 88% (Ormrod and Miller, 1980; Giacomini et al., 1981) of renal tissue. The study of Konrad and Husberg (1984) reported that the animals were usually clinically ill by 3 months. Hostetter et al. (1986) have recently 88 P. D. Acott et al. reported a similar technique in a smaller group of animals who were then subdivided to receive very low or very high protein intakes. They noted that a very high protein intake may accelerate the progression of glomerulosclerosis and increase BUN and proteinuria. No animals were followed into terminal uremia, and it was not possible to relate protein Chronic dition feeding renal failure under the influence rats in this study followed model potential to have a period decline w o rk c ulture s Me d ic a l kind ly C he m istry of uremia in their sample. of compensation in renal function, can be expected con- models, after as occurs to survive exposure as a model evolving the aforementioned renal after the injury many renal for many months, of chemicals of slowly progressive it is as is intended uremia in gives it much fields of study. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ w a s sup p o rte d w e re C linic a l Its uniqueness in other Da lho usie Unlike for the study of long-term this laboratory. This appeared in man. As many animals an ideal progression is not a static state but a complex of many factors. by an exponential insults the to the biochemical in humans b y MRC Re se a rc h G ra nt 7640. 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