Diabetes Research and Clinical Practice 24 (1994) 161-165

The effect of pancreatic elastase on diabetic nephropathy

Takashi Nagai

The First Department of Internal Medicine. Gumna University School of Medicine, 3-39-15 Showa Machi, Maebashi, Gunma, 371, Japan

(Received 2 August 1993; revision received 27 December 1993; accepted 22 February 1994)

Ahstract

Pancreatic elastase has been shown to inhibit the thickening of glomerular basement membranes in experimental diabetic animals. We explored the clinical significance of the prolonged administration of pancreatic elastase on diabetic nephropathy, in patients whose blood glucose levels and blood pressure were controlled. Pancreatic elastase was administered for 12 months. Body weight levels, blood glucose levels, HbA,, values and blood pressure remained unchanged. Administrationof 10 800 U of pancreatic elastase caused a significant decrease in albuminuria (before ad- ministration, 512.4 f 79.8 mg/g creatinine vs. 12 months after administration, 284.1 + 61.9 mg/g creatinine, P < 0.01, n = 28). In the contrast group (n = 18), no significant changes in albuminuria were observed after administration of 300 mg of dilazep dihydrochloride. Serum levels of creatinine and urinary levels of NAG and &MG were not affected by pancreatic elastase. The present study indicates a significant inhibitory effect of pancreatic elastase on increased albuminuria in diabetic patients.

Keywords: Pancreatic elastase; Diabetic nephropathy; Urinary albumin; Urinary N-acetyl-&Dglucosaminidase; Urinary &-microglobulin

1. IntrlMIuctIon

Diabetic nephropathy is mainly due to a micro- angiopathy characterized by proteinuria and an inevitable decline in the glomerular filtration rate. It is a major cause of mortality in diabetic patients. Diabetic glomerular lesion is characterized by a thickening of the glomerular basement membrane and an increase in collagen-like substances within the mesangial regions [ 1,2]. Although blood glu- cose and blood pressure are important factors in the control of diabetic nephropathy, the two fac-

tors alone may be insufficient in slowing the course of diabetic nephropathy. Fabre et al. reported that 40-60% of the cases of diabetic patients whose blood glucose and blood pressure were fully con- trolled still have excessive urinary protein excre- tion [3]. A strong and sensitive predictor of the development of diabetic nephropathy is the evalu- ation of elevated rates of urinary albumin excre- tion [4,5]. Pancreatic elastase is an elastin lytic enzyme [6], which has a collagenolytic action in liver cirrhosis [7], and has shown an inhibitory effect on the thickening of the glomerular base-

0168-8227/94/%07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-8227(94)00879-Y

162 T. Nagai/ Diabetes Res. C/in. Pracr. 24 (1994) 161-165

ment membrane in experimental diabetic animals [8,9]. In this paper, we report on the inhibitory effect of pancreatic elastase on nephropathy in diabetic patients whose blood glucose and blood pressure remained stable. We administered dilazep dihydrochloride to the contrast diabetic group. This drug is known as an anti-platelet agent [lo], which has been used against glomerulonephritis with the aim of reducing proteinuria and preven- ting progression of the disease [ 10, Ill.

2. Subjects, materials and methods

2. I. Subjects

Forty six (24 men and 22 women) non-insulin- dependent diabetic patients diagnosed more than 10 years previously, took part in this study (Table 1). All patients showed diabetic retinopathy, and all urinary specimens showed f to 2+ proteinuria and negative for hematuria. All patients were on a diabetic diet including a protein intake of 1.0 to 1.2 g per kg body weight. Twenty patients were given an oral hypoglycemic drug (sulfonylurea). In 22 patients, insulin treatment was given for the control of blood glucose. Hypertension was found in 31 of the 46 patients, and was normalized or partially corrected by the administration of anti-

hypertensive drugs, the dose and type of which was not changed from two years prior to the study.

2.2. Renal functions Creatinine (Cr) was measured by Jaffe’s rate

assay. Urinary samples were collected in the early morning. Urinary albumin was measured by means of latex turbidimetric immunoassay [ 121. N- Acetyl-&Dglucosaminidase (NAG) was measured using the fluorocytometry method. Latex photo- metric immunoassay was used to measure &- microglobulin (&MG). The urinary albumin index (Alb-I) was calculated as urinary albumin/ urinary Cr (mg/g Cr). Also, the urinary NAG index (NAG-I) or urinary &MG index (&MG-I) was calculated as urinary NAG/urinary Cr (units/g Cr) or urinary &MGlurinary Cr (&g Cr).

2.3. Method

Patients who had f to 2+ proteinuria, showed more than 150 mg/g Cr of Alb-I in the 7 months prior to the study. They were given daily oral doses of either 10 800 U of pancreatic elastase (Eisai Co. Ltd., Tokyo, Japan 113,141) (group A, n = 28) or 300 mg of dilazep dihydrochloride (Kowa Co. Ltd., Nagoya, Japan [lo]), (group B, n = 18) for 12 months. Measurements were taken

Table I Changes in various parameters before and after the administration of pancreatic elastase or dilazep dihydrochloride

Group A (n = 28)

Before 12 months after

Group B (n = 18)

Before 12 months after

Age (years) 62.9 zt 1.6 63.9 zt 1.6 63.1 jz 2.2 64.1 f 2.2 BMI (kglm2) 22.3 ztz 0.4 22.4 * 0.4 22.2 * 0.4 22.2 f 0.5 FBG (nimoi/I) 6.8 * 0.2 7.0 f 0.2 6.6 f 0.2 6.7 f 0.3 HbA,, (%) 8.0 f 0.1 8.0 f 0.2 7.8 f 0.1 8.1 f 0.2 sBP (mmHg) 146 f 2 140 f 4 146 zt 2 143 l 2 dBP (mmHg) 81 l I 79 f 3 82 +z 2 80 zt 3 T-ch (mmolil) 5.4 f 0.1 5.1 f 0.1 5.2 * 0.1 5.2 f 0.2 Cr (PmoliI) 80.1 zt 3.3 82.6 * 3.5 79.1 * 4.6 82.4 zt 5.9 Alb-I (mg/g Cr) 512.4 f 79.8 284.1 f 61.9’ 521.4 f 98.9 428.9 f 112.2 NAG-I (units/g Cr) 18.1 zt 1.6 18.0 ztz 2.0 19.0 f 2.0 18.3 zt 2.7 &MG-I (r&g Cr) 1240.1 f 340.9 1056.1 f 298.2 1239.2 f 378.6 1050.1 f 298.9

Group A: diabetic patients given pancreatic elastase. Group B: diabetic patients given dilazep dihydrochloride. l P < 0.01 (before V.S. 12 months after in each group).

T. Nagai / Diabetes Res. Clin. Pratt. 24 (1994) 161-165 163

at 2,4,6,9 and 12 months after the administration of each drug.

2.4. Statistical analysis Parameters were shown as mean f S.E.M. The

data in each group were analyzed by Duncan’s multiple range test and the data between two groups were analyzed every other month by the Student’s t-test.

and the total cholesterol levels remained unchang- ed during the study in both group A and group B. Table 1 and Fig. 2 show changes in Alb-I after administration of pancreatic elastase or dilazep dihydrochloride. Significant reduction in Alb-I occurred 2 months after administration of pancre- atic elastase (before administration, 512.4 i 79.8 mg/g Cr vs. 2 months after administration, 275.1 * 54.7, P < 0.001). The pancreatic elastase- induced decreases in Alb-I were sustained over 12 months (P c 0.01). In contrast, treatment with dilazep dihydrochloride did not cause a significant change in Alb-I (P > 0.05). Serum levels of Cr and NAG-I and &MG-I were neither affected by pan- creatic elastase or dilazep dihydrochloride treat- ment.

3. Results

As shown in Table 1 and Fig. 1, mean body mass index, fasting blood glucose levels, HbAi,, systolic blood pressure, diastolic blood pressure

pancreatic elastase 10800 u/day

dilazep dihydrochloride 300&day

I

systolic BP

3 140 S-S- L c P

L __ diastolic BP

I N I I I I 1 I

or-1 0 2 4 6 I

9 12

month (s)

Fig. 1. Changes in various background parameters after the administration of pancreatic elastase (-O-) or dilazep dihydro- chloride (-O-).

2 90.0

t z 80.0

600

200

2 20

2 - dJ

16

$

3 1600

5 1200

p 600 s

pancreatic elastase 10800 u/day or dilarep dihydrochloride 3OOwr/dt

I

I I I 1

-10 2 4 6 9 12 month (s)

Fig. 2. Changes in various renal function parameters after administration of pancreatic elastase (-O-) or dilazep dihydro- chloride (-O-). *P < 0.01 (before V.S. 2, 4, 6, 9 or 12 months after the administration of each drug).

164 T. Nagai /Diabetes Res. Clin. Pratt. 24 (1994) 161-165

There were no significant differences in values such as Cr, Alb-I, NAG-I and &MG-I between the two groups at bimonthly analyses.

No abnormalities were noted in general labora- tory findings including hematology and chemistry during a 1Zmonth period of treatment with pan- creatic elastasc.

4. DIsc&on

Available data have shown the value of correc- ting blood glucose [ 151, hypertension [ 161 and urinary infection [18] in an effort to improve the course of diabetic nephropathy. However, it has been reported that 40% of 510 diabetic patients with satisfactory blood control still had excessive urinary protein excretion and that 58% of the patients with strictly normal blood pressure had obvious proteinuria [31. The pathological change of diabetic nephropathy is characterized by a thickening of the glomerular basement membrane [ 1,2]. Elastase is a pancreatic enzyme that hydrolyzes elastin and regulates the metabolism of elastin in arterial walls and connective tissues [6]. A decrease in elastase appears to disturb the metabolism of vascular elastic fibers [ 171. Pancre- atic elastase has shown an inhibitory effect on the thickening of the glomerular basement membrane in experimental diabetic animals [7,8]. The present results show that the administration of pancreatic elastase significantly reduces albuminuria in diabetic patients, while no significant reduction of albuminuria occurs in diabetic patients treated with dilazep dihydrochloride. During the course of the study, control of body weight, blood glucose, HbAi, and blood pressure remained unchanged. Therefore, decreased albuminuria after the admin- istration of pancreatic elastase was not the result of changes in these factors. The mechanisms underlying elastase-induced decreases in albumin- uria remains unknown. Pancreatic elastase did not affect urinary NAG and urinary &MG levels while the drug was being administered. Urinary NAG, which is a lysosomal glycoprotein- degrading enzyme of proximal tubules [19], has been shown to be a marker for subtle renal tubular injury [20]. Urinary &MG is an indicator of renal tubular function since it is filtered and reabsorbed

in the side of proximal tubules [21]. In conjunction with these observations, the present data may in- dicate that pancreatic elastase may not act on the proximal tubules, but on the glomerular basement membrane.

Acknowledgements

The authors would like to thank Professor Masatomo Mori, of the First Department of Inter- nal Medicine, Gunma University School of Medi- cine, for valuable discussions.

Referemxs

1

2

3

4

5

6

I

8

Osterby, R. (1973) A quantitative electron microscopic study of mesangial lesions in glomeruh from patients with short term juvenile diabetes mellitus. Lab. Invest. 29, 268-370. Spiro, R.G. (1963) Glycoproteins and diabetes. Diabetes 12.223-230. Fabre, J., Balant, L.P., Dayer, P.G., Fox, H.M. and Vemet, A.T. (1982) The kidney in maturity-onset diabetes mellitus: a clinical study of 510 patients. Kidney Int. 21, 730-138. Mogensen, C.E. (1984) Microalbuminuria predicts clini- cal proteinuria and early mortality in matu.rity-onset diabetes. N. Engl. J. Med. 310, 356-360. Mogensen, C.E. and Christensen, C.K. (1984) Predicting diabetic nephropathy in insulin-dependent patients. N. Et@. J. Med. 311, 89-93. Balo, J. and Banga, 1. (1950) The elastolytic activity of pancreatic extracts. Biochem. J. 46, 384-387. Fukuhara, M. (1975) Inhibition of experimental liver fi- brosis by Ccl,, following treatment with elastase. J. Nara. Med. Ass. 26, 198-209. Oikawa, S., Kakizaki, M. and Goto, Y. (1982) Inhibitory effect of pancreatic elastase on thickening of the renal glomerular basement membrane in the spontaneous diabetic rat. Tohoku J. Exp. Med. 138, 103-109. Shibata, M., Kawanishi, A., K&hi, T. et al. (1981) In- hibitory effect of elastase on the glomerular capillary basement membrane thickening of the experimental con- genital diabetic mice (N.S.Y. mice). Nagoya J. Med. Sci. 43, 111-115. Mitsumasa, N., Shuzo, K., Kenji, S. and Nishio, H. (1985) Amelioration of albuminuria induced by dilazep adminis- tration in aminonucleoside nephrosis of rats. Jap. 1. Nephrol. 21, 385-391. Hiromi, I., Akio, K., Mitsuharu, N. and Shizuo, T. (1985) Effects of the antiplatelet agents, dipyridamole and dilazep dihydrochloride on in vivo function and pro- teinuria. Jap. J. Nephro. 27, 1261-1270. Shuichi, I., Morifinni, W., Toshiyuki, Y. et al. (1987)

T. Nagai/Diabetes Rex Clin. Pratt. 24 (1994) 161-165 165

Latex turbidiietric immunoassay with LA-system for urinary albumin. J J C L A 12, 144-148 (in Japanese).

13 Atsushi, K. and Masayuki, Y. (1981) Crystallization and identiAcation of a binary complex of a elastase-I and a carboxypeptidase AT from porcine pancreas. Biochem. Biophys. Res. Commun. 100, 1091-1098.

14 Takeyuki, K., Teruaki, Y., Jiro, H. and Eiji, I. (1991) Concentratiion of hog elastase I in serum of healthy sub- jects after oral administration measured by sensitive two- site enxyme immunoassay. Clin. Chem. Enxym. Commun. 4, 123-133.

15 Viberti, G.C., Pickup, J.C., Phil, D., Jarrett, R.J. and Keen, H. (1979) Effect of control blood glucose on urinary excretion of albumin and & microglobulin in insulin-dependent diabetes. N. Engl. J. Med. 300, 638-641.

16 Mogensen, C.E. (1976) Progression of nephropathy in long-term diabetics with proteinuria and effect of initial anti-hypertensive treatment. Stand. J. Clin. Lab. Invest. 36. 383-388.

17 Vejlsgaard, R. (1966) Studies on urinary infection in diabetes. Significant bacteriuria in relation to long-term diabetic manifestations. Acta. Med. Stand. 179, 183- 188.

18 Balo, J. and Banga, I. (1953) Change in the elastase con- tent of the human pancreas in relation to arteriosclerosis. Acta. Physiol. Acad. Sci. Hung. 4, 187-194.

19 Lehis, M., Dubaeh, V.C. and Schmidt, U. (1979) Quanti- tative distribution of lysosomal hydrolases in the rat nephron. Histochemistry 63, 254-251.

20 Kunin, C.M., Chesney, R.W., Craig, W.A., England, AC. and DeAngelis, C. (1978) Enzymuria as a marker of renal injury and disease: studies of N-acetyl-beta- glucosaminidase in the general population and in patients with renal disease. Pediatrics 62, 751-760.

21 Peterson, P.A., Evrin, P.E. and Berggard, I. (1969) Differ- entiation of glomerular, tubular and normal proteimuia: determinations of urinary excretion of &microglobulin, albumin, and total protein. J. Clin. Invest. 48, 1189-I 198.