The effect of intraperitoneal administration of Phlogenzym on the
development of accelerated glomerulosclerosis in Goldblatt
hypertensive rats
Šebeková K.1, Heidland A.2 , Krivošíková Z.,
Daemmrich J.
1 Clinic of Pharmcotherapy, Institute of Preventive and
Clinical Medicine Bratislava, Slovakia
2 Medizinische Klini, University of Wuerzburg,
Wuerzburg, Germany
Study No.:MU-697411
Report on an experimental in vivo study, June 23, 1997
Introduction
Hypertensive nephrosclerosis is one of the most important causes of
the end-stage renal failure. The contralateral, non-clipped kidney,
of two-kidney one-clip (2K1C) Goldblatt renovascular hypertensive
rats is used as a model of hypertensive nephrosclerosis. Except for
the hypertensive damage of glomeruli, other mechanisms, such as cell
proliferation, participation of mononuclear cells (lymphocytes and
macrophages) at extraglomerular site, and accumulation of
extracellular matrix molecules participate during the development of
hypertensive damage. Though, except for glomeruli, the tubules and
the interstitium are actively involved. The contribution of the
altered protease activities in the development of hypertensive renal
injury is well documented, however, the exact role of the growth
factors and cytokines still remains to be elucidated.
Recently, evidence was given, that oral enzyme treatment may have
beneficial effects even in non-immune mediated diseases. Our last
data in the rat model of subtotal nephrectomy indicated, that
systemic treatment with proteases retards the progression of renal
disease. Therefore, we raised the question, whether enzyme treatment
might be beneficial in the retardation of the renal injury in the
Goldblatt hypertensive rats.
Aim of the study
Primary aim
The primary study tasks were:
to investigate the effect of a 7 weeks long intraperitoneal
administration of Phlogenzym® on the development of hypertensive
glomerulosclerosis in Goldblatt hypertensive rats,
to evaluate renal histology of the non-clipped kidney at the end of
the study.
Secondary aims
to study the effect of Phlogenzym® treatment on the development of
renal hypertension
to confirm, whether i.p. administration of Phlogenzym® might exert a
catabolic effect in this model of renal damage (employing the
pair-feeding of the animals)
to investigate the effect of i.p. administration of Phlogenzym® on
standard biochemical parameters, acid-base balance, and blood
picture
to investigate the effect of systemic treatment with proteases in
the Goldblatt rats on the activities of proteases in isolated
glomeruli and tubules
in studies of the role of growth factors, adhesion molecules and
cytokines in the development of hypertensive nephrosclerosis the
following investigations should be done:
- evaluation of the effect of Phlogenzym® administration on the
renal excretion of TGF-
- in the case the standard histology shows beneficial effect of the
treatment, the staining of the deep-frozen renal tissues for
adhesion molecules, growth factors and cytokines should be performed
to elucidate their participation in the course of the disease and
during the treatment
since chronic renal insufficiency induces hypertrophy of the heart
and damage of the heart muscle in subtotally nephrectomized rats.
Specimen of heart will be preserved into formalin as well as by
deep-freezing for potential evaluation of the changes induced during
renal hypertension, and the effect of the treatment might be
evaluated
as rutosid in Phlogenzym® acts as a scavenger, the total antioxidant
status (TAS), gluthation peroxidase (GPX) as well as superoxid
dismutase (SOD) activities determination at the end of the study
might be of importance, elucidating the potential ratio in isolated
glomeruli and tubules may have changed, therefore the expression of
the activities per mg DNA, as well as the protein/DNA ratio changes
are expected to be conclusive.
Conclusions
Tolerability and Toxicity
I.p. administration of Phlogenzym to Goldblatt hypertensive
rats was well tolerated without visible toxicity.
Physical Parameters
I.p. administration of the enzymes exerts slight catabolic
effect. Lower heart weight of the enzyme treated rats in spite of
the high blood pressure might point to the prevention of cardiac
hypertrophy.
Blood Chemistry
Trends towards lower serum creatinine and urea concentration
were observed in the enzyme treated Goldblatt hypertensive rats in
comparison to the placebo group.
Renal Function
As a consequence of renal injury serum creatinine tended to
higher levels, while serum urea increased significantly, and
proteinuria was higher, too. Enzyme treatment prevented the rise in
proteinuria. Trends towards lower serum creatinine and urea
concentration, as well as higher creatinine and urea clearance were
observed in the enzyme treated Goldblatt hypertensive rats in
comparison to the placebo group.
Morphological Analysis
57% of the untreated animals with renovascular hypertension
developed the classical changes of malignant nephrosclerosis, which
particularly involved the small renal arteries. Tubulointerstitial
expansion was associated with infiltration of monocytes/macrophages
and T-helper cells. The interstitial volume index increased by a
factor of two.
Discussion
Summarising, our data demonstrate, that sustained systemic treatment
with proteases in the model of 2K1C renovascular hypertension in
rats ameliorates the severity of nephrosclerosis and
tubulointerstitial fibrosis, although the rise in blood pressure
remained unaffected. It is assumed that the administered proteolytic
enzymes induce the proteolytic effects by cleavage of adhesion
molecules and increased clearance of growth promoters and cytokines
due to their binding to an activated a2-protease complex. Therefore,
proteases may modulate the various amplifying mechanisms involved in
the progressive kidney damage. For more detailed discussion see the
submitted manuscript.