Impaired renal function in diabetic chimpanzees (Pan troglodytes)

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

38,224-229

(1983)

Impaired Renal Function in Diabetic Chimpanzees (Pan troglodytes) I.Y. White

Sands

Received

ROSENBLUM' AND F. COULSTON

Research Center, Alamogordo, June

Colt&on International New Mexico 88310

25, 1982, and in revised

form

September

Corporation,

13, 1982

Studies were conducted to assess the renal functional state in two recently discovered diabetic chimpanzees. Both were nonobese, adult female animals with the non-insulindependent form of impaired glucose tolerance, analogous to the Type II or nonobese, maturity-onset diabetes of humans. Both animals displayed moderate-to-heavy proteinuria and glycosuria in response to intravenous administration of glucose or tolbutamide. Chimpanzee number 333, but not number 1037, had fasting proteinuria and chronic hypertension. Renal function studies, using the inulin clearance method, demonstrated signifkantly decreased glomerular filtration rates and elevated rates of sodium excretion for both animals. The rate of chloride excretion was also elevated in animal number 1037, but potassium excretion was apparently unaffected in both animals. Abnormal serum biochemical parameters demonstrated for chimpanzee number 333 included elevations in calcium, magnesium, creatinine, urea nitrogen, and uric acid; animal number 1037 had only an elevated serum creatinine. Results are consistent with the occurence of renal disease similar to the nephropathy that develops in human diabetics. The difference in severity of renal impairment in the two chimpanzees is possibly related to differences in duration and severity of impaired glucose tolerance. A progression of both diabetic and renal disorders is most probable.

INTRODUCTION Glucose intolerance, indicative of overt diabetes mellitus, was recently reported in four adult chimpanzees (Rosenblum el al., 1981). On the basis of biochemical and histological studies, these animals were classified as “Impaired Glucose Tolerant,” analogous to the Type II, non-insulin-dependent diabetes of humans. Clinical diagnostic tests and routine monitoring revealed varying degrees of proteinuria in two of the four diabetic animals, suggesting the occurrence of renal disease. Since nephropathy is a frequent pathological complication of diabetes mellitus, further studies were conducted to define the nature of the impaired renal function in these two chimpanzees. MATERIALS

AND METHODS General methods and procedures. Data on two diabetic and six nondiabetic (control) chimpanzees are presented in Table I. All animals were housed in indoor-outdoor cages and maintained on Purina Jumbo Biscuit Chow (Ralston Purina Co., St. Louis, MO.), supplemented three times a week with fresh fruit, and tap water. All animal manipulations and tests were performed following sedation with Ketoset (Ketamine HCl, Bristol Laboratories, Syracuse, N.Y.). Plasma was obtained from blood samples collected in EDTA tubes (Becton-Dickinson, Rutherford, N.J.); serum was taken from separate blood samples after clotting and centrifugation. Blood samples for biochemical determinations were routinely drawn between 0800 and 0900 hr subsequent to an 18-hr overnight fast. r Current address: Philadelphia College of Pharmacy and Science, 43rd St. and Kingsessing Mall, Philadelphia, Pa. 19104. To whom correspondence and reprint requests should be sent. 224 0014~4800/83 $3.00 Copyright @ 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

IMPAIRED

RENAL FUNCTION

IN DIABETIC

TABLE I Vital Statistics of Experimental

CHIMPANZEES

225

Subjects

Animal number

Sex

Age (yd

Weight (kg)

Diabetic0

333 1037

F F

24 21

41 50

Nondiabetic controls

753 781 870 940 1035 1040

F F F F F F

10 8 7 7 8 7

43 29 36 27 34 29

Classification

n Non-insulin-dependent,

analogous to Type II nonobese maturity-onset

human diabetic.

Analytical methods. Serum levels of urea nitrogen, creatinine, albumin, total protein, glucose, and uric acid were routinely analyzed using an automated centrifugal analyzer system (Centrifmhem 300 Series, Union Carbide Corp., Pleasantville, N.Y.). All other tests were conducted manually. Sodium and potassium levels were determined by flame photometric procedures, chloride by a modiflcation of the Oxford titration method, and calcium by a fluorescent titration method (Tie& 1976). Magnesium was determined by atomic absorption spectrometry (Hansen and Freier, 1967), glucose by the hexokinase method (Sonowane et al., 1976), urea nitrogen by the urease method (Bretaudiere et al., 1978), creatinine by the Jatfe reaction (Fabiny and Ertingshausen, 1978), uric acid by a modified uricase method (Pesce et al., 1974), albumin by the bromcresol green reaction, and total protein by the biuret reaction (Savory et al., 1978). Quantitative calorimetric procedures were used to measure serum levels of total cholesterol (Ellefson and Caraway, 1976), triglycerides (Van Handel and Zilversmit, 1957), and total lipids (Bragdon, 1960). Plasma and urine concentrations of inulin were quantitated using a modification of the method of Heyrovsky (1956). Urinalyses were performed using reagent strips based upon semiquantitative calorimetric reactions (Bio Dynamics, Indianapolis, Ind.). The method used for estimating total urine protein concentration was based on the quantitative determination of urine albumin and globulin fractions using an electrophoretic separation procedure (Anderson et al., 1979). Renal clearance measurements. Standard renal clearance techniques were used. Glomerular filtration rate (GFR) was estimated from the renal clearance rate of inulin (C,,) according to procedures previously established in the chimpanzee (Fanelli et al., 1971). A 10% stock solution of pyrogen-free inulin (CalbiochemBehring, San Diego, Calif.) was freshly prepared and sterilized prior to each experiment. Solutions to be infused were always equilibrated at 37°C and passed through coiled tubing immersed in a constant-temperature water bath. Priming injections of inulin (50 mg/kg) were followed by constant intravenous infusions at the rate of 1.5 mg/kg/min (3 ml/min) using a Harvard variable-speed peristaltic pump (Harvard Apparatus Co., South Natick, Mass.). After an equilibration period of 1 hr, accurately timed 30-m% urine collection samples were secured together with heparinized femoral artery blood samples taken at the midpoint of each period. Baseline and postinfusion samples included both clotted and hepa-

ROSENBLUM

226

AND COULSTON

rinized bloods (Becton-Dickinson). Foley catheters (General Medical, El Paso, Tex.) were used for urine collection. Lactated Ringer’s solution (Abbott Laboratories, North Chicago, Ill.) was used as the inulin diluent and the vehicle for the baseline infusion periods. Statistics. Standard statistical methods were used to evaluate the resultant data. The Student t test was performed to determine significant differences between compared values. RESULTS

AND DISCUSSION

Results of the inulin clearance experiments are summarized in Table II. Compared to six nondiabetic control animals, both diabetic animals displayed abnormally low rates of giomerular filtration and significantly high sodium excretion rates. Chimpanzee number 1037 also had an elevated rate of chloride excretion compared to controls. Potassium excretion rates were within the normal range for both diabetic animals. Selected serum biochemical values for the diabetic and control chimpanzees are summarized in Table III. Compared to the nondiabetic controls, diabetic number 333 had significantly increased serum values of calcium, magnesium, creatinine, urea nitrogen, and uric acid. Serum creatinine was significantly elevated in diabetic number 1037, but this animal also had significantly decreased values of serum potassium and chloride when compared to the nondiabetic controls. With the exception of serum triglyceride concentration, which was elevated in animal number 333, serum lipid measurements were within the normal range of values established for stock chimpanzees at this facility. Recently, glucose intolerance, indicative of overt diabetes mellitus, was documented in four chimpanzees (Rosenblum et al., 1981). Since that report, two of these apes died and the remaining two (numbers 333 and 1037) are the subjects of this report. Glucose clearance rates, as well as the plasma responses of insulin, C-peptide, and pancreatic glucagon, were analyzed from a series of intravenous glucose and tolbutamide tolerance tests. The results demonstrated that all of these parameters were impaired in animals numbered 333 and 1037 compared to nondiabetic, healthy controls. Significant declines in glucose clearance rates (K,, %/min) were observed: number 333, 0.87 ? 0.01; number 1037, 1.44 2 0.44; controls, 2.72 2 0.47. Insulin and C-peptide responses were markedly deficient, and glucagon levels were consistently elevated compared to controls. Chimpanzee number 333 showed no measurable change in plasma glucose or insulin following TABLE II Results of Inulin Clearance Experiments in Two Diabetic and Six Nondiabetic Control Chimpanzees Parametel”

Cm W-W Sodium excretion Chloride excretion Potassium excretion

Units

Diabetic 333 (n = 11)

Diabetic 1037 (n = 11)

Controls (n = 20)

ml/mm/kg peq/min peqlmin peqlmin

0.70 2 0.13’ 269 2 296 26O-t29 69 f 6

1.29 2 0. 14d 253 k 146 347 + 2ff 91*7

1.94 2 0.22 193 2 26 257 ” 37 83 ?I 10

o Values represent means f SE; n = number of experimental observations. b Glomerular filtration rate estimated as the inulin clearance rate, C,,. c Significantly different from controls, P < 0.001. d Significantly different from controls, P < 0.05.

IMPAIRED

RENAL FUNCTION

IN DIABETIC

CHIMPANZEES

227

TABLE III Comparison of Serum Biochemical Values In Diabetic and Control Chimpanzees Serum

analysis

Sodium Potassium Chloride Calcium Magnesium Glucose Creatinine Urea nitrogen Uric acid Total protein Albumin Total cholesterol Total lipids Triglycerides

Units

w@wq/L wdL mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl 01 g/d1 mg/dl mg/dl mg/dl

Diabetic 337 137 f 1 3.4 f 0.2 103 25 4.9 f 0.T 2.3 f 02 91 ?6 3.4 f 0.4r 31 *Y 5.1 f 0.6r 7.0 f 0.10 3.1 2 0.1 234 k 27 486 f 116 358 T 670

Diabetic 1037 138 f 3.0 f 102 * 4.2 f 1.8 t 93 f 1.7 + 19 f 4.7 f 6.9 + 3.6 f 237 k 566 f 212 +

2 02 1” 0.1 0.0 F 02 5 1.1 0.5 0.1 64 146 48

Nondiabetic controlsc 138 3.8 108 4.3 1.9

+ 1 f 0.1 &2 2 0.1 f 0.0 0.8 f 0.1 20 f 2 2.8 + 0.3

-

Normal rms+ 138 I 1 3.7 f 0.1 105 k 1 4.4 ” 0.1 1.9 2 0.1 76 k 6 0.9 f 0.0 13 f 1 3.6 f 0.2 6.6 f 0.1 3.5 f 0.1 234 + 11 301 It: 31 200*12

a Mean + SE of five determinations. b Mean f SE of four determinations. c Mean + SE, n = 6. Na, K, and Cl determinations in triplicate; all others in duplicate. d Normal range of values determined for stock adult chimpanzees; mean + SE, n = 32 (21 males and 11 females). Values analyzed in triplicate except: glucose, n = 10; calcium, n = 22; magnesium, n = 20; and the serum lipid fractions, n = 12. es’ Values statistically different compared to controls; e P < 0.05; f P < 0.081. 0 Values statistically different from normal range, P < 0.05.

tolbutamide stimulation, results which further support the diagnosis of overt diabetes. The glucose intolerance appeared to be less severe in chimpanzee number 1037. Following stimulation with tolbutamide, a time-dependent hypoglycemic response occurred in spite of the fact that plasma insulin levels remained unchanged (Rosenblum et al., 1981). Historically, proteinuria is one of the oldest known signs of diabetic nephropathy. In a rat model of experimental diabetes, it was reported that total urinary protein excretion increased relative to increased duration of diabetes (Weber et al., 1979). Changes in renal protein clearance, as well as increased renal capillary permeability, have been demonstrated in human diabetics (Jet-urns et al., 1973; Alpert et al., 1972). A recent statistical study of risk factors for nephropathy in diabetic humans revealed that the duration of diabetes was strongly related to the development of heavy, but not mild, proteinuria (West et al., 1980). In the same study, it was shown that frequency of proteinuria (of any degree) was not strongly related to the duration of diabetes, but was related to the level of fasting plasma glucose at the time of examination. Etiology of diabetes in these two chimpanzees remains uncertain. The nephropathy is presumed, but not proven, to be related to the impaired glucose tolerance. Animal number 333, born in captivity, had been pregnant at least seven times; two offspring were raised, but five other offspring were stillborn. Signs of hypertension, hyperglycemia, and proteinuria were recorded in 1976 just prior to, and following, her fifth stillbirth (D. Hill and 3. Bailey, personal communication). The administration of an intravenous glucose tolerance test, approximately 1 month postpartum, showed mild glucose intolerance which suggested the possibility of

228

ROSENBLUM

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COULSTON

gestational diabetes (D. Hill and J. Bailey, personal communication). There was no follow-up until late 1978, when further testing revealed chronic hypertension and mild-to-moderate proteinuria and glycosuria. Results of clinical thyroid tests were negative. Urine protein analysis revealed total protein excretion in excess of 1 g/24 hr. Albumin excretion was significantly elevated. Chimpanzee number 1037, also born in captivity, had never been pregnant. Fasting blood glucose levels were marginally elevated but, unlike chimpanzee number 333, urinary protein excretion was within the normal range (cl50 mg/24 hr). However, the occurrence of proteinuria and glycosuria was repeatedly demonstrated in response to intravenous glucose and tolbutamide tolerance tests. Both diabetic chimpanzees were nonketotic and nonobese. According to recently proposed human diagnostic criteria, they are analogous to a Type II, maturity-onset human diabetic (National Diabetes Data Group, 1979). Although both animals are non-insulin dependent at this stage, it is highly probable that their disease will progress to the stage of insulin requirement. Based on the known pathogenesis and clinical sequela of diabetes mellitus in humans and in animals, a progressive severity of nephropathy can be anticipated, as well as the development of other, e.g., vascular, complications. The pathogenic role of hypertension in animal number 333 also requires further definition. Recent ocular examinations have revealed no apparent pathological changes in the eyes of either animal. The sexual cycles of both females appear regular; nevertheless, various breeding attempts since 1978 have been unsuccessful. Finally, these findings represent the first detailed documentation of a nephropathic disorder in the great apes that resembles the diabetic nephropathy seen in man. Not only do the data stress the value of continued monitoring of nonhuman primate species, they reinforce the use of chimpanzees as human surrogates for selective biomedical investigation. Identification and characterization of the various disease states in the chimpanzee should further our understanding of the corresponding human disorders. ACKNOWLEDGMENTS The authors wish to acknowledge Brenda Billhymer.

the skilled

technical

assistance

of Ms.

Rose Normand

and Ms.

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