© 1991 S. Karger AG. Basel 0250-8095/91/0115-0386S2.75/0

Am J Nephrol 1991;11:386-390

Urinary Kallikrein Excretion in Chronic Pancreatic Diseases1 Carlo Fabris3, Maria Piera Panozzob, Daniela Bassoc, Giuseppe Del Faverob, Mario PlebanF, Martina Zaninottoc, Paola Fogarb, Tamara Meggiatob, Paola Scalonb, Chiara Ferrarab, Remo Naccaratob “Cattedra di Medicina Interna, Universitá degli Studi di Udine; b Istituto di Medicina Interna (Cattedra di Malattie Apparato Digerente);c Istituto di Medicina di Laboratorio, Universitá dcgli Studi di Padova, Italy

Key Words. Urinary kallikrein • Pancreatic ribonuclease • Pancreatitis • Tubular damage Abstract. Variations in urinary kallikrein in pancreatic diseases were ascertained, and possible influencing factors were investigated. Serum amylase and urinary excretion of glandular kallikrein, pancreatic ribonuclease (RNase), y-glutamyltransferase (GGT) and amylase were measured in 24 control subjects, 39 patients with pancreatic cancer, 49 with pancreatitis and 63 with extra-pancreatic diseases. Urinary kallikrein was found to be elevated in a substan­ tial number of patients with pancreatitis. Higher levels were detected in patients with a relapse, which was diagnosed using clinical and biochemical examinations. RNase was also increased in a high number of patients with pancreatic diseases, but was not correlated with pancreatic damage. In patients with pancreatitis, a correlation was found between urinary kallikrein and RNase excretions. No correlations were found between kallikrein and serum or urinary amylase and GGT. We can conclude that urinary kallikrein excretion increases in pancreatitis, especially when a phlogistic involvement of the pancreas is present; this condition may lead to a release of this ultrafiltrable enzyme in the circulation. Renal tubular damage, which determines a reduced reabsorption of this enzyme, seems to play a concomitant but minor role in this process.

The kallikrein-kinin system is involved in several pathways of renal physiology and pathophysiology [1-3]. In particular, urinary kallikrein seems to regulate, at least in part, renal blood flow and electrolyte excretion [2-7], An association has been found between decreased urinary kallikrein excretion and hypertension and so­ dium retention. [5, 8, 9]. Most urinary kallikrein is probably produced in the kidney [10, 11]. However, the pancreatic exocrine gland is one of the other possible sources of this ultrafiltrable enzyme. Therefore, in patients with pancreatic diseases, variations in glandular kallikrein excretion may be ex­ pected. 1 Supported in part by a grant from the Centro Regionale di Alta Specializzazione per lo Studio delle Malattie del Fegato e del Pan­ creas. Under the auspices of the ‘R. Farini Association for Gastroen­ terological Research’.

The aim of this study was to ascertain whether there are any modifications in the urinary kallikrein excretion of patients with chronic pancreatic disease, comparing them with healthy and pathological controls, and also to study any factors that might be responsible for such modifications. Materials and Methods This study comprised 175 subjects: 39 patients had pancreatic cancer of duct cell origin (21 males. 18 females, age range 2 8 -8 1 years) that was histologically confirmed [ 12] by means of surgical or autoptic specimens; 49 had pancreatitis (5 acute and 44 chronic; 41 males, 8 females, age range 26-70 years). The diagnosis of acute pancreatitis was based on clinical, biochemical and radiological findings. Chronic pancreatitis was diagnosed on the basis of the clinical picture and positive findings from at least 2 of the following: plain abdomen X-ray, US, CT and/or ERCP. 27 of the patients with chronic pancreatitis were studied during a clinical relapse. 63 patients had extra-pancreatic diseases mainly of gastrointestinal ori­ gin (40 males, 23 females, age range 23-77 years). Diagnoses were

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based on the clinical picture and on findings from specific radiolog­ ical and histological investigations: primary liver cell cancer (2 cases), carcinoma of the hepatic hilus (1), gastric cancer (2), carci­ noma of the esophagus (1), bile duct cancer (3), carcinoma of the ampulla of Vater (4), carcinoma of the colon (2), gallbladder carci­ noma (1), lymphoma (1), liver cirrhosis (15), chronic hepatitis (5), diverticuiosis of the colon (2), duodenal ulcer (2), Crohn’s disease (2), liver angioma (1), irritable colon (4), benign stenosis of the papilla of Vater (2), celiac disease (2), polyp of the colon (1), gall­ stones (6), portal hypertension (I), chronic cholangitis (2), hiatus hernia (1). The control group consisted of 24 healthy members of the med­ ical staff or blood donors (16 males, 8 females, age range 23-43 years). No patient had a history of overt renal disease. All patients underwent renal function tests. All had normal serum creatinine levels. No hematuria, pyuria, bacteriuria or frank glomerular pro­ teinuria ( > 1.0 g/day) were found. Urinary kallikrein, RNase and GGT were measured in dialized urine by means of previously reported methods [13-16]. Serum and urine amylase were assayed using Rauscher’s method [ 17], A statistical evaluation of the results was made by means of the analysis of variance (Anova one way), Bonferroni’s test for pairwise comparisons [18] and Student’s t test. Where appropriate, a loga­ rithmic transformation of the data was performed before making the analyses.

Results

Fig. 1. Individual values of urinary kallikrein excretion in the material studied. Continuous line represents the upper normal limit (mean ± 2 SD of our controls). CS = Control subjects, PC = pan­ creatic cancer; P = pancreatitis; EPM » extra-pancreatic malignan­ cies; EPBD = extra-pancreatic benign diseases; A = acute pancreati­ tis; o = chronic pancreatitis studied during a relapse. Anova one way: F = 5.16, p < 0.001; Bonferroni’s test for pair­ wise comparisons: * p < 0.005, as compared to PC. Fig. 2. Individual values of urinary ribonucléase (RNase) excre­ tion in the material studied. The continuous line represents the upper normal limit (mean + 2 SD of our controls). CS = Control subjects; PC = pancreatic cancer; P = pancreatitis; EPM = extrapancreatic malignancies; EPBD = extra-pancreatic benign diseases; A = acute pancreatitis; o -= chronic pancreatitis studied during a relapse. Anova one way (logarithmically transformed values): F = 3.11, p < 0.05; Bonferroni’s test for pairwise comparisons: * p < 0.01, as compared to PC, P and EPBD, and p < 0.05 as compared to EPM.

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Figure 1 shows the individual values and a statistical analysis for urinary kallikrein excretion in our cases. Fig­ ure 2 shows the urinary excretion and the results of a statistical analysis of RNase in the subjects considered. Figure 3 illustrates the individual values of serum and urinary amylase in our patients.

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Fabris/Panozzo/Basso/Del Favero/Plebani/Zaninotto/Fogar/Meggiato/Scalon/Ferrara/Naccarato

AM YLASE

U /L

U R IN A R Y A M Y L A S E mU/min-NT’

In patients with pancreatic cancer, no significant dif­ ference was found between those with and those without liver métastasés for urinary kallikrein outputs (t = 1.309, p = n.s.). In patients with pancreatitis, a higher mean kallikrein output was found in the presence of an acute or relapsed disease (t = 2.352, p < 0.025). Furthermore, pancreatitis patients with hyperamylasemia or hypergly­ cemia had more elevated kallikrein outputs than those with normal serum amylase or glucose (t = 2.076, p < 0.025, t = 1.944, p < 0.05, respectively). The above vari­ ables caused no significant variation in urinary ribonu­ cléase values. Overall, no significant correlation was found between urinary kallikrein and (1) urinary RNase (r = 0.106, p = n.s.); (2) GGT (r = 0.077, p = n.s.); (3) serum (r = -0.012, p = n.s.), and (4) urinary amylase (r = -0.035, p = n.s.). On the other hand, in patients with pancreatitis, a signif­ icant correlation was found between urinary kallikrein and urinary RNase (r = 0.391, p < 0.01). No correlations were found in any patients between urinary kallikrein and (1) serum ALP (r = -0.023, p = n.s.) or (2) total bilirubin (r = -0.038, p = n.s.).

Discussion The amino acid composition and therefore the molec­ ular mass of glandular kallikreins, a group of serine pro­ teases [19] present in a variety of tissues [20-23], are

strikingly similar [22, 24], Glandular kallikreins act on both low and high molecular weight kininogens, leading to the formation of kinins. In a number of studies, it has been observed that urinary and pancreatic kallikreins are analogous [10, 11, 22, 24], Glandular kallikrein is a light protein (molecular weight 25,000 D) and, like other low molecular weight proteins, undergoes renal ultrafiltra­ tion and active tubular reabsorption which is a highcapacity but low-affinity transport process. Therefore, only a small fraction of the ultrafiltered enzyme escapes in urine [25, 26]. In this study, a substantial number of patients with pancreatitis had an increased urinary kallikrein output. This enzyme was also found to be occasionally elevated in patients with pancreatic cancer and in a number of patients with biliary tract diseases. Serum RNase is another low molecular weight pro­ tein produced by several tissues, such as glandular kalli­ krein. However, the major sources of this enzyme are the pancreas and the kidney [27], RNase increases in serum in the course of several diseases, especially in the pres­ ence of neoplasia [28]. An increased RNase urinary excretion was found in most of the patients with pan­ creatic diseases, although sometimes high levels were found in patients with other gastrointestinal diseases. The elevated urinary levels of both enzymes may depend upon their augmented release into the blood­ stream during pancreatic inflammation and damage. This occurs in pancreatitis but also in the presence of

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SERU M

Fig. 3. Individual values of serum and uri­ nary amylase in the material studied. PC = Pancreatic cancer; P = pancreatitis; EPM = extra-pancreatic malignancies; EPBD = extrapancreatic benign diseases; o = acute pancre­ atitis and relapsed chronic pancreatitis; A = benign liver biliary diseases.

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krein excretion. Therefore, in patients with pancreatic diseases, tubular damage seems to play a less important role than pancreatic phlogosis in determining an in­ creased kallikreinuria.

References 1 Levinsky NG: The renal kailikrein-kinin system. Cire Res 1979; 44:441-451. 2 Carretero OA, Scicli AG: The renal kallikrein-kinin system. Am J Physiol 1980;238:F247-F255. 3 Margolius HS: The kallikrein-kinin system and the kidney. Annu Rev Physiol 1984;46:309-326. 4 Margolius HS, Horwitz D, Geller RG, et al: Urinary kallikrein excretion in normal man. Circ Res 1974;35:812-819. 5 Koolen MI. Daha MR, Van Brumniclcn P: Is the renal kallikrein system relevant to sodium sensitivity in patients with essential hypertension? Eur J Clin Invest 1985;15:151-156. 6 Cumming AD, Jeffrey S, Lambie AT, et al: The kallikrein-kinin and renin-angiotensin system in nephrotic syndrome. Nephron 1989;51:185-191. 7 Waller DG, Bhatia SS, Campbell SK, et al: Active and inactive urinary kallikrein in man: Effects of diuresis and antidiuresis. ClinSci 1990;79:117-121. 8 Margolius HS, Horwitz D, Pisano JJ, et al: Urinary' kallikrein excretion in hypertensive man. Relationship to sodium intake and sodium-retaining steroid. Circ Res 1974;35:820-825. 9 Mersey JH, Williams GH, Emanuel R, et al: Plasma bradykinin levels and urinary kallikrein excretion in normal renin essential hypertension. J Clin Endocrinol Metab 1979;48:642-647. 10 Fink E, Amouric M, Geiger R, et al: Comparison of immunolog­ ical and enzymatic properties of human urinary and pancreatic kallikrein. Hoppe-Seylcr’s Z Physiol Chem 1982:363:819-823. 11 Fink E, Schleuning M: Studies on the excretion of tissue kalli­ krein from blood into the urine. Hoppc-Seyler’s Z Physiol Chem 1982;363:1331-1339. 12 Cubilla AL, Fitzgerald PJ: Pancreas cancer. 1. Duct adenocarci­ noma. Pathol Annu 1978:13:241-289. 13 Meani A. Fabris C, Vianello D, et al: A kinetic assay for human urinary kallikrein determination. Enzyme 1985;33:89-93. 14 Reddi KK. Holland JF: Elevated serum ribonucléase in patients with pancreatic cancer. Proc Natl Acad Sci USA 1976;73:2308— 2310. 15 Szasz G: A kinetic photometric method for serum gamma-glutamyltransfcrasc. Clin Chem 1969;15:124-136. 16 Fabris C. Meani A. Farini R, et al: Urinary ribonucléase excre­ tion in pancreatic disease. J Clin Chem Clin Biochem 1983;21: 573-575. 17 Rauscher E, Neumann U, Schaich E, et al: Optimized conditions for determining activity concentration of amylase in serum, with l,4-a-D-4-nitrophcnylmaltoheptaoside as substrate. Clin Chem 1985;31:14-19. 18 Wallenstein S, Zucker LC. Fleiss JL: Some statistical methods useful in circulation research. Circ Res 1980:47:1. 19 Geiger R. Fritz H: Human urinary kallikrein. Methods Enzymol 1981:80:466-493. 20 Geiger R. Stuckstedtc U, Fritz H: Isolation and characterization

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pancreatic cancer and biliary diseases, pathologies in which a synchronous pancreatic involvement may be present, as shown by the hyperamylasemia observed in some of these patients. As it is well known that jaundice and liver damage may cause enzymuria, it has been sug­ gested that pancreatic phlogosis may also alter renal tubular function and cause cellular tubular damage, pos­ sibly through the release of nephrotoxic substances (pro­ teolytic enzymes?) [29, 30], In a previous study, we dem­ onstrated that there is a link between the severity of pan­ creatic inflammation assessed by serum pancreatic en­ zymes and the degree of tubular dysfunction [31]. How­ ever, apart from the nature of the damaging factor, renal tubular impairment may cause an increase in the excre­ tion of both urinary kallikrein and RNase by reducing their reabsorption. Since both enzymes could also be of renal origin, their increase in urine may be related to their increased release by damaged tubular cells. Urinary kallikrein was found to be more closely asso­ ciated to the degree of pancreatic phlogosis than to other parameters; this can be explained by the action of glan­ dular kallikrein in phlogosis. Our patients with acute pancreatitis and relapsed chronic pancreatitis had higher kallikrein values than patients with quiescent disease. Furthermore, hyperamylasemia was linked to higher kal­ likrein outputs. Hyperglycemia, which may be related in chronic pancreatitis to a more severe and/or to a relaps­ ing disease, was also correlated with increased urinary kallikrein excretion. On the other hand, the spread of pancreatic tumor was not found to influence urinary kal­ likrein excretion. Urinary RNase excretion was not found to be related to pancreatic phlogosis. Further­ more, the spread of pancreatic tumor did not influence urinary RNase outputs, suggesting that multiple factors, apart from the presence of pancreatic damage, are in­ volved in increasing urinary RNase. These observations stress the different pathophysiological conditions related to variations in kallikrein and RNase levels in the circu­ lation and in urine. Several urinary enzymes have been investigated in order to assess the presence and degree of a tubular dam­ age. Two major classes have been identified: indices of tubular functional impairment (including RNase), and high molecular weight enzymes indicators of anatomical tubular damage (including GGT). Overall, in our pa­ tients, no correlation was found between urinary kalli­ krein and RNase or GGT. In pancreatitis, a relationship was found between kallikrein excretion and RNase. Fi­ nally, no correlation was found between the presence of cholestasis, a nephrotoxic condition, and urinary kalli­

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Fabris/Panozzo/Basso/Dcl Favero/Plebani/Zaninotto/Fogar/Meggiato/Scalon/Ferrara/Naccarato

of human urinary kallikrein. Hoppe-Seyler’s Z Physiol Chem 1980;361:1003-1016. Geiger R, Clausnitzer B: Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography. Hoppe-Seyler’s Z Physiol Chem 1981 ;362: 1279-1283. Hofmann W, Geiger R: Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice. Hoppe-Seyler’s Z Physiol Chem 1983:364:413-423. Hofmann W, Junk A, Geiger R: Human tissue kallikrein. II. Isolation and characterization of human salivary kallikrein. Hoppe-Seyler’s Z Physiol Chem 1983;364:425-432. Lottspeich F, Geiger R, Henschen A, et al: N-terminal amino acid sequence of human urinary kallikrein homology with other serine proteases. Hoppe-Seyler’s Z Physiol Chem 1979;360: 1947-1950. Maack T, Johnson V, Kau ST, et al: Renal filtration, transport, and metabolism of low-molecular-weight proteins: A review. Kidney Int 1979;16:251-270. Sumpio BE. Hayslett JP: Renal handling of proteins in normal and disease states. Q J Med (New Series) 1985;57:611-635. Neuwelt EA, Boguski MS. Frank JJ, et al: Possible sites of origin of human plasma ribonuclease as evidenced by isolation and partial characterization of ribonuclease from several human tis­ sues. Cancer Res 1978;38:88-93.

28 Basso D, Fabris C, Meani A, et al: Serum deoxyribonuclease and ribonuclease in pancreatic cancer and chronic pancreatitis. Tumori 1985:71:529-532. 29 Meier PB, Levitt MD: Urine protein excretion in acute pancre­ atitis. J Lab Clin Med 1986:108:628-634. 30 Mock DM, Grendell JH, Cello J, et al: Pancreatitis and alcohol­ ism disorder the renal tubule and impair reclamation of some low molecular weight proteins. Gastroenterology 1987:92:161 — 170. 31 Fabris C, Basso D, Del Favero G, et al: Renal tubular dysfunc­ tion in pancreatic cancer and chronic pancreatitis. Nephron 1989;51:56-60.

Received: February 20. 1991 Accepted: August 7, 1991 Prof. Remo Naccarato Istituto di Medicina Interna Cattedra di Malattie Apparato Digerente Via Giustiniani 2 1-35128 Padova (Italy)

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Urinary kallikrein excretion in chronic pancreatic diseases.

Variations in urinary kallikrein in pancreatic diseases were ascertained, and possible influencing factors were investigated. Serum amylase and urinar...
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