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Effects of Protease Therapy in the Remnant Kidney model of Progressive Renal Failure.

K Sebekova^a, L. Paczek^b, J. Dämmrich^c, H. Ling^d, V. Spustova^a, Z. Gaciong^b, and A. Heidland^d. Effects of Protease Therapy in the Remnant Kidney model of Progressive Renal Failure. Mineral and Electrolyte Metabol. 1997: 23, pp 291-295. 344 KA (17-05-1).
a Institute of Preventive and Clinical Medicine, Bratislava, Slowakia; b The Transplantation Institute Warsaw, Poland; c Institute of Pathology; d Department of Internal Medicine, University of Wuerzburg, Germany.

Abstract
This study investigated whether protease treatment ameliorates the progressive course of chronic failure in the rat model of subtotal nephrectomy. Fourteen male Wistar rats underwent 5/6 nephrectomy, and were randomized into a control group (C, n = 7) given 2 ml of 0.9% NaCl intraperitoneally (i.p.) daily, and a study group (P, n=7) treated with 12 mg Phlogenzym^® (combination of trypsin, bromelain and rutosid) in 2 ml saline i.p. daily. After 6 weeks treatment, the Phlogenzym group showed lower proteinuria (C:19.6±9.1 vs 10.2±6.2 mg/24 h, p < 0.05). Endogenous creatinine clearance was higher (C: 192.3 ±99.4, P: 300.5 ±47.9 ml/min per 100 g, p < 0.05), while plasma creatinin was decreased (C: 106.7 ± 33.9, P: 76.0 ± 6.3 mmol/l, p < 0.01). Blood urea nitrogen levels did not change, although urea clearance tended to be higher in the protease-treated rats. Decreased renal formation of cytokines was reflected by a lower urinary excretion ratio of transforming growth factor (TGF)-b/creatinine (C: 0.363 ±0.183, P: 0.232 ±0.085 ng TGF-b/mg creatinine, p < 0.05). Renal morphology revealed less infiltration of mononuclear cells and an amelioration of interstitial fibrosis as expressed by the volume index of the cortical region (C: 17.17 ± 1.43; P: 12.3 ±0.5%, p < 0.001). In addition, the activities of lysosomal proteinases (cathepsin B, L + B, and H), which are decreased in the remnant kidney model of chronic renal failure, were significantly higher in the enzyme-treated group both in isolated glomeruli and proximal tubules. The body and kidney weight tended to be lower, probably due to a catabolic action of the enzymes. In summary, we provide evidence that protease treatment may be beneficial in a nonimmune mediated renal disease. Phlogenzym ameliorated the course of chronic renal failure in the rat model of subtotal nephrectomy and retarded the development of tubulointerstitial fibrosis. Decreased cytokine formation in the remnant kidney is supposed to play a key role.
Key Words: Interstitial fibrosis, proteases, remnant kidney

Introduction
Proteases are of fundamental importance in cellular and extracellular protein turnover in the glomeruli [1, 2]. In various models of chronic renal diseases, proteolytic activity in isolated glomeruli and tubules is markedly reduced [3-9]. Such a decrease was demonstrated both by employing the azocasein test and by determining various proteases (cathepsin B, L + B, and H, as well as collagenase and gelatinase). Moreover, inhibition of the plasmin protease system has been found in glomerulosclerosis [10-12]. As a consequence of impaired protease activities, cellular hypertrophy and accumulation of extracellular matrix in glomeruli and tubulointerstitial tissue may occur [12].
It was recently shown that systemic administration of proteases ameliorates various models of immune-mediated glomerulonephritis [13, 14; E.J. McKelvey et al., submitted]. Furthermore, administration of proteolytic enzymes exerted salutary effects on the accelerated arteriosclerosis of aortic allografts [15]. Up to now there are no data available whether protease treatment improves non-immune-mediated renal diseases. We therefore investigated whether sustained administration of Phlogenzym (a combination of trypsin, bromelain, and rutosid) ameliorates the progressive course of chronic renal insufficiency in the rat remnant kidney model.

Material and Methods
The study was approved by the Institutional Ethics Committee for Animals in Bratislava.
Animal
Fourteen male Wistar rats (Velas, Praha, Czech Republic) weighing 180-220 g undewent 5/6 nephrectomy (NX), according to Morrison [16]. They were fed a standard rat chow in the form of pellets with a protein content of 20%; drinking water was provided ad libitum.
Experimental Protocol
Starting from the second day after 5/6 NX, 2 ml of 0.9% NaCl solution were given intraperitoneally to the control group (n = 7) while 12 mg of Phlogenzym (trypsin 2.5 mg, bromelain 4.59 mg, and rutosid 5.1 mg; Mucos Pharma, Geretsried, Germany) in 2 ml of 0.9% NaCl were administered to the study group (n = 7) daily. A day before sacrifice, each animal was placed into a metabolic cage designed for quantitative urine collection.
Biochemical Analysis
Blood. Plasma levels of electrolytes, creatinine, urea, uric acid, glucose, total proteins, cholesterol, and triglycerides were determined employing a Kodak Ektachem 700 analyser (Rochester, N.Y USA).
Urine. Analysis was performed for proteinuria (Biuret method), creatinine, and urea (Kodak Ektachem 700 analyser), N-acetyl-(l-D-glucosaminidase ((b-NAG) activity (Hitachi 911 analyser, Boehringer Mannheim, Germany), and transforming growth factor-b (TGF-b) (Elisa Kit Prodictaâ , Genzyme, Cambridge, Mass., USA).
Kidneys. Glomeruli and proximal tubules were isolated by a differential sieving technique [17] for determination of protein [18], DNA [19] and lysosomal activities of cathepsin B, L + B and H [20] in the cell lysates.
Renal Histology
Renal tissue of the remnant kidney was fixed in formalin and embedded in paraffin. Tissue sections (4 mm thick) were stained with hematoxylin and eosin (HE), periodic acid-Schiff, and PAMS. A double-blind evaluation was performed by a pathologist using light microscopy. The percent volume fraction of renal interstitial tissue (Vi%) in the cortical region was calculated with a point counting method: ten square test fields (100 intersection points, 0.16 mm² test field area) engraved on the ocular were evaluated using a magnification of 400 [21].
Statistics
Results are given as the mean and 95% confidence limits (95% CL). The Wilcoxon test for unpaired samples was used to compare the means between the groups (p < 0.05 was considered significant).
Results
Physical Parameters
After 6 weeks, the body and kidney weight of the protease-treated group tended to be lower (table 1). The kidney/body weight ratio was identical in both groups.
Blood Pressure
At sacrifice, the blood pressure of normal control rats averaged 114.2 ± 5.5 mm Hg. In the NX rats, the blood pressure rose to 148.0 ± 8.6 mm Hg in the placebo-treated group and to 146.3 ± 7.5 mm Hg in the enzyme treated rats.

Table 1. Effects of protease treatment on body and kidney weights in 5/6 NX rats

Table 2. Effect of protease therapy on blood chemistry in 5/6 NX animals

Blood Chemistry
The mean values of plasma electrolytes (Na, K, Ca, Mg) did not differ between the groups (data not given). A trend for lower concentrations of total protein and cholesterol as well as higher levels of triglyceride and glucose was observed in the enzyme-treated group (table 2).
Renal Function
Diuresis tended to be lower in the enzyme-treated rats, while proteinuria was reduced. Plasma creatinine was also significantly less due to a higher creatinine clearance (table 3). Urea clearance tended to be higher in the protease-treated animals, although the mean plasma urea level was unchanged (table 3). The urinary excretion ratio of TGF-b/creatinine was lower in the actively treated group, while lysosomal cathepsin (B, L + B, and H) activities both in tubules and glomeruli were higher during protease treatment (table 4). Urinary activity of b-NAG did not differ between the groups.

Table 3. Effects of protease therapy on parameters of renal function

Table 4. Activities of lysosomal proteases (cathepsin B, L+B, and H; mmol/ml per minute per microgram DNA) in isolated glomeruli and tubules of subtotally nephrectomized rats after 6 weeks of treatment

Renal Morphology
In the NaCl-treated 5/6 NX rats, the interstitium of the cortical tissue was increased with focal accentuation around small arterial vessels and arterioles. The amount of collagen fibers was enhanced and the number of infiltrating mononuclear cells, especially lymphocytes and macrophages, was clearly increased (fig. la). These alterations were markedly less in the protease-treated animals (fig. 1b). Morphometrically, the Vi% in renal cortex was significantly elevated in untreated 5/6 NX rats (17.2± 1.4%) compared to the protease-treated rats ( 12.3 ± 0.5%, P < 0.01 ), in which the mean value was only slightly higher than normal values (9.3 ± 0.3%).
Tolerance and Toxicity
Intraperitoneal administration of Phlogenzym to the 5/6 NX rats in a dose of 12 mg/day seemed to be safe: no signs of intolerance were observed. One rat administered saline died after 4 weeks, while all enzyme-treated rats survived.

Discussion
The remnant kidney model induced by 5/6-nephrectomy is characterized by a hypertrophic response of the residual nephrons with a time-dependent development of glomerulosclerosis and tubulointerstitial fibrosis. The glomerular protein/DNA ratio is enhanced, while cysteine and metalloproteinases are impaired both in isolated glomeruli and proximal tubules [9]. These lowered activities may be mediated by the action of TGF-b, which increases the tissue levels of the specific matrix metalloproteinase inhibitors [21] and the plasminogen activator inhibitor-1 [10]; it decreases the synthesis of metalloproteinases [21] and impairs the activity of cathepsins [22]. In various models of glomerulosclerosis and tubulointerstitial fibrosis, including the remnant kidney, overproduction of this cytokine results in cellular hypertrophy and an increase in extracellular matrix [23].
In the present study, daily intraperitoneal administration of proteases to 5/6 NX rats elevated cathepsin activity both in isolated glomeruli and tubules, with an associated marked improvement of the tubulointerstitial fibrosis. The volume fraction of cortical tissue, proteinuria and urinary excretion ratio of TGF-b/creatinine decreased while creatinine clearance increased significantly. If the urinary levels of TGF-b reflect the concentration of the active component of this cytokine in renal tissue, both the enhanced cathepsin levels as well as the amelioration of tubulointerstitial fibrosis could be explained by this alteration.
In contrast to the decline of plasma creatinine concentrations, blood urea levels did not change in the protease-treated 5/6 NX rats, although the clearance of urea tended to be higher. This dissociation may be in part due to a decline of diuresis in the protease-treated group which favors the backdiffusion of urea along the nephron. Furthermore, the unchanged blood urea concentration despite enhanced creatinine clearance may be a consequence of a catabolic action of Phlogenzym. In line with this assumption is the finding that the protease-treated group showed an insignificant decline in body weight as well as a slightly lowered total protein and cholesterol concentration in the plasma while blood glucose levels tended to be higher. Observed by our group [Sebekova et al., unpublished data] under strict pair-feeding conditions in a renewed experiment in NX rats as well as in Goldblatt hypertensive rats, underlining the catabolic properties of intraperitoneal administered hydrolytic enzymes.
How can the beneficial action of protease therapy in the remnant kidney model be explained? It may be argued that systemic administration of proteases cannot be of any value because they will bind to protease inhibitors such as a₂macroglobulin (a2M) and a₁-antitrypsin (a1-AT). However, binding of proteases to a2M to the amide bonds differs from that of the classic inhibitor a1-AT [24]. Thus, the protease active sites are not totally blocked resulting in persistent proteolytic activity at least for reactions with smaller sized proteins. Furthermore, protease binding is followed by an activation of native a2M to its fast form which generates new binding sites at the thiol ester for cytokines and growth factors [25]. Thiol ester activation of a2M enhances binding of TGF-b, platelet-derived growth factor, fibroblast growth factor and interleukin-1b [26]. In addition, the protease-induced structural changes of a2M generate new binding sites for specific a2M receptors, resulting in an enhanced clearance of these cytokines [27]. As a consequence, cytokine and growth factor levels may be lowered in the damaged tissue, allowing an improvement of abnormal cell growth and cell proliferation and reduced extracellular matrix formation in the remnant kidney model [28].
Another potential effect of protease therapy may be a selective effect on cellular adhesion molecules. Thus in vitro and in vivo studies have shown that trypsin cleaves the adhesion molecules CD44, CD4, and B7-1 of activated T cells and macrophages [29]. Bromelain, the other component of the employed enzyme preparation, also cleaves CD44 and may thereby enhance the response to trypsin [30]. As a consequence of these effects, activation of T lymphocytes and macrophages in the interstitium, which mediate the inflammatory response, could be down-regulated.
Finally, the administered proteases trypsin and bromelain may activate various matrix metalloproteinases which are synthesized and secreted as inactive zymogens. Following limited proteolysis of the amino terminus, they become actively proteolytic. This has been demonstrated in in vitro studies [1] and in recent in vivo investigations, in which intravenous administration of trypsin to unilateral NX rats activated the latent collagenases MMP2 and MMP9 in the renal tissue [3l]. The salutary effect of Phlogenzym therapy was not caused by a lowering of arterial hypertension since the blood pressure was identical in the treated and non-treated NX rats.
In summary, the data demonstrate that intraperitoneal administration of proteases in the remnant kidney model ameliorates the severity of tubulointerstitial fibrosis. It is assumed that the salutary actions of administered proteolytic enzymes result from the combined effects of inactivation and enhanced removal of growth promoters and cytokines (following their binding to an activated a2M-protease complex), cleavage of various adhesion molecules and restoration of some impaired proteolytic activities, thereby ameliorating various amplificatory mechanisms involved in the renal damage in 5/6 NX rats.

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This study was also published in other journals, which have the following reviews on this website::
Inter. Journal of Tissue Reactions 1997, Vol. XIX, No. 1/2, pp 97 -  abstract
Nieren-und Hochdruckkrankheiten, 1997, Jahrgang 26, Nr. 6, pp. 277 - 281; 345 KA (5-08-1)