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HEXA
M I N I M A L I S T P R E S E N TAT I O N
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I n t r o d u c ti o n

• Acute Kidney Injury (AKI) mortality rates remain quite high, despite improved strategies
for supporting vital organs during AKI recovery and in renal replacement therapy
(dialysis) (Leite et al. 2013)

• Red Propolis has been classified as a separate type based on its unique chemical
composition, particurlaly rich in isoflavonoids (Right et al. 2013). Anti-inflammatory and
antioxidant properties have been attributed to red propolis (Bueno-Silva et al. 2013;
Enis Yonar et al. 2012)

Methods
• Animals and Red Propolis
Wistar rats, weighing 250-300g,
maintained under controlled
temperature (21 +- 2o C) and humidity
conditions (60 +- 5%) with a 12:12-h
light : dark cycle. A standart commercial
pellet diet and water offered.
Red Propolis was collected in mangrove
region in Marechal Deodoro (a city in
the northeastern Brazil).
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• Chemical characterization of red
propolis
40 grams of propolis macerated in 100ml
of 40% ethanol, for 30 days -> solution
filtered -> filtrate evaporated to dryness
-> residue 12,5 g of dry extract ->
dissolved in 50 ml of ethanol with
concentration 0,25g/ml -> HPLC (high-
performance liquid chromatography)
The chromatographic method shows
linearity over the range evaluated and the
correlation coefficients for formononetin
and biochanin A were 0,9915 and 0,9996.
The concentrations of formononetin and
biochanin A in the propolis extracted were
10.25 +- 0,21 and 0,50 +- 0,02 μ g/mg,
respectively.

Methods
• Surgical Procedure
Animals were anesthetized with sodium
pentobarbital (50 mg/kg).
The right kidney was removed and left
ischemic renal failure was induced by
clamping the renal artery (with a
nontraumatic clamp) for 60 min,
followed by reperfusion.
After 48h, animals were sacrificed to
obtain blood samples for biochemical
tests. Left kidney were collected for
histological and immunohistochemistry
evaluation.
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• Experimental Groups
Rats were divided into the following groups :
 Sham + tap water group (SHAM)
Rats -> identical surgical procedures, except for
the nephrectomy and unilateral renal occlusion
shock, kept anesthesia.
 Sham + red propolis (RP)
Rats -> same with SHAM, receiving red propolis
(150 mg/kg/day) -> administred by gastric
gavage before procedure.
 I/R + tap water group (IR)
Rats -> submitted to nephrectomy and
unilateral renal occlusion (60 min) followed by
reperfusion
 I/R + red propolis group (IR-RP)
Rats -> above mentioned surgical procedure
and red propolis -> gastric gavage -> and
ischemia
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Methods

• Measurement of biochemical
parameters

48h after ischemia, rats were

reanesthetized, and blood samples
(1ml) were collected via venipuncture.
The samples were centrifuged
(6000rpm for 3 min) to separate
plasma.

Plasma and urine concentration of urea

(BUN) and creatinine (Cr) -> indicators
of impaired glomerular function.
Plasma and urine concentration of
sodium (Na+) and potassium (K+) ->
indicators of renal turbular injury.

Measured by colorimetric methods in a


Methods
• Determination of MDA levels
Malondialdehyde (MDA) concentration in kidney
tissue was determined as an indicator of lipid
peroxidation. Left kidney homogenized with KCl
to make homogenate -> solution containing
phosporic, thioarbituric acid added -> mixture
heated in boiling water -> n-butanol added, and
shaken -> absorbance measured by
spectrophotometry at 532. Result expressed in
nmol/g tissue
• Determination of GSA levels
Homogenate was prepared with EDTA -> added
distillated water and tricloroacetic acid ->
centrifuged 3000rpm 15min -> supernatant
collected -> Tris-HCL buffer, DTNB added ->
measured by spectro at 412 nm. The
concentration of gluthathione was expressed in
micrograms per gram of tissue.
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• Histological analyses
Renal tissuewas removed and placed in
a10% solution of buffered formaldehyde
-> after 24h, the tissue was transferred to
an alcoholic solution (70%) and used for
histological analysis.
• Immunohistochemical localization
of heme-oxygenase and eNOS
(Endothelial NOS)
Performed using streptavidin-biotin-
peroxidase method.
• Western Blot analysis
To detect specific proteins in a sample of
tissue homogenate or extract.

Results
• Red Propolis attenuates I/R-
induced functional impairment
Animals submitted to I/R Injury had an
increment in serum CR and urea,
reflecting a marked reduction in CrCl.
Red propolis attenuated the functional
alterations induced by I/R injury.
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• Effects of red propolis on histologic
injury
Light microscopy studies showed tubular
necrosis, tubular dilation, inflammatory
cell infiltration and cellular edema in the
tubular interstitium from animals.
These lesions were less intense in rats
where treated with red propolis when
compared with untreated animals.
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Results

• Red Propolis reduced I/R-induced

oxidative stress

After red propolis treatment , there was

a significant decrease in
Malondialdehide (MDA) levels after I/R
Injury. The values of GSH were
significantly improved by the red
propolis treatment
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Results

• Red Propolis restores the I/R

induced downregulation of eNOS
48hours after surgical procedures, the
IR group rats showed markedly lower
eNOS protein expressions.

Red Propolis administration attenuated

the down regulation of eNOS
expression, as can be seen in Fig. 5,
confirmed by western blot analysis Fig.
6.
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Results

• Heme Oxygenase-1 upregulation

by red propolis

Heme oxygenase-1 immunostaining in

the IRRP group was significantly
increased when compared with IR
group. Although the western blot
analysis disclosed similar HO-1
expression in SHAM and IRG Groups, it
confirmed the increased in the IRRPG
group.
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