The effects of plant-derived oleanolic acid on kidney function in male Sprague-Dawley rats and, in cell lines of the kidney and liver.
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2012
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Abstract
Adverse effects and increasing cost of therapeutic drugs have renewed an interest in the use of
medicinal plant products for the treatment of a variety of chronic disorders. One such bioactive
plant-derived compound is a pentacyclic triterpenoid, oleanolic acid (3ß-hydroxy-olea-12-en-28-
oic acid, OA) present in herbs. OA possesses a variety of pharmaceutical activities and of
interest in this study are the anti-diabetic properties. Diabetes is associated with disorders
grouped as microvascular (retinopathy and nephropathy) and macrovascular (atherosclerotic)
complications. Accordingly, this study further investigated the potential of OA in diabetes
management by studying the effects of this triterpene on kidney function as well as proximal
tubular Na+ handling in an effort to identify the site of action of OA. Furthermore, the study
evaluated the effects of OA in kidney and liver cell lines to establish whether this triterpene
exhibits any toxicity in these organs.
OA was extracted using a previously validated protocol in our laboratory. Briefly, dried flower
buds of Syzygium aromaticum were soaked in dichloromethane overnight, thereafter in ethyl acetate to
obtain ethyl acetate solubles which contained a mixture of OA/ursolic and maslinic acid (MA). OA/MA
mixture was subjected to column chromatograph and pure OA was obtained through recrystallization in
methanol. The absolute stereostructure of OA was elucidated using 1H and 13C NMR spectroscopy and
was comparable to previously reported data. In kidney function studies, various doses of OA (30, 60,
120 mg/kg, p.o.) were administered to male Sprague-Dawley rats twice (8h apart) every third day
for five weeks. Rats administered deionised water served as controls. Measurements of body
weight, food and water intake, blood pressure, Na+, K+, Cl-, urea and creatinine were taken 24 h
from dosing. Renal clearance studies investigated the influence of OA on Na+ handling in the
proximal tubule of anaesthetized rats using lithium clearance. Animals were given water with
lithium (12mmol/l) for 48 hours following which they were anaesthetized and cannulated using a
previously validated standard protocol that has been reported from our laboratories. After a 3½ h
equilibration, animals were challenged with hypotonic saline for 4 h of 1 h control, 1½ h
treatment and 1½ h recovery periods. OA was added to the infusate during the treatment period.
In vitro effects of various OA concentrations (5, 10, 20, 40, 80 μmol/l) were investigated in
HEK293, MDBK and HepG2cell lines. Cells were exposed to OA for 24, 48 and 72 h, thereafter,
3-4,5 dimethylthiazol-2-yl- 2,5diphenyltetrozolium bromide (MTT) and single cell gel
electrophoresis (comet) assays were conducted. All data are presented as means ±SEM.
OA significantly (p<0.05) increased urinary Na+ output from week 2 until the end of the
experimental period in a dose independent manner. However, this OA-evoked natriuresis was not
reflected in plasma collected at the end of the experiment as there was no change in plasma Na+
concentrations compared with control animals at the corresponding time. OA administration had
no significant influence on K+ and Cl- excretion rates throughout the experiment. However, OA
significantly (p<0.05) reduced plasma creatinine concentration with a concomitant increase in
glomerular filtration rate (GFR). Furthermore, OA administration significantly (p<0.05)
decreased mean arterial pressure from week 2 until the end of the experimental period.
Intravenous infusion of OA at 90 ug/h for 1 ½ h induced a marked increase in urinary excretion
rates of Na+. This increase was accompanied by concomitant increase in FENa proximal and FENa
distal and FELi which persisted until the end of the experiment without any apparent changes in
GFR. The cell viabilities of HepG2, HEK293 and MDBK cell lines were significantly increased
after 24 h exposure, however, the viabilities of all the three cell lines dropped after 72 h exposure
to values that did not achieve statistical significance in comparison to the respective controls. In
addition, all OA-treated cells in the comet assay had intact DNA after exposure for 24, 48 and 72
h. Hence, the decrease in viability that was observed in the MTT assay after 72 h exposure could
probably be attributed to the depletion of nutrients in the culture medium.
The results of the present study, apart from confirming our previous observations of the
natriuretic effects of OA in rats, indicate that this effect is in part mediated via the inhibition of
proximal tubular Na+ reabsorption and increased Na+ secretion. We speculate that this increased
Na+ secretion could have been due to increased tubular function and not to the toxicity of OA as
indicated by MTT and comet assays. These findings suggest that OA does not exhibit toxicity in
the kidney and the liver.
Description
Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.
Keywords
Kidney function tests., Medicinal plants--Analysis., Theses--Human physiology.