Swiss albino male mice weighing 25±g were used for the experiment. These animals were acclimated to 22±1°C and were maintained under 12-h periods of light and dark each, with free access to clean water and commercial mice food. The animals were housed in polypropylene cages inside a well-ventilated room.

Mice were randomly distributed into three groups, each containing 10 mice. Group 1 mice received saline and served as control group while group 2 mice received intraperitoneal injection of gentamicin at dose of 80mg/kg for 7 consecutive days and this group was marked as Gm group. Mice in group 3 were treated as group 2 and were additionally co-administered with 500mg/kg of propolis one hour-post gentamicin injection and this group was marked as GmP group.

Following treatments as described above, each mouse was weighed; kidneys were removed and weighed. Finally, the kidney index was calculated by dividing the left kidney weight by the body weight and then multiplying by 100 and the results were statistically analyzed by SPSS software (SPSS Inc.).

Blood samples for the measurement of blood chemistry were drawn into prechilled tubes containing EDTA, and immediately placed on ice. Serum in the samples was separated by centrifugation at 3000rpm and stored at −80°C until assay. Serums were used for the estimation of blood urea nitrogen (BUN) and creatinine.

Paraffin embedded kidney sections were deparaffinized in xylene and rehydrated in descending grades of alcohol and finally distilled water. Sections were then heated in citrate buffer (pH 6) in microwave for 5min, washed with PBS buffer for 5min and were incubated in peroxidase blocking solution for 10min. After blocking sections were incubated overnight at 4°C with diluted primary antibody (anti-caspase3 ab13585, anti-Kim-1, rabbit polyclonal antibody ab78494, anti-malondialdehyde ab194225). Sections were then incubated with biotinylated goat anti-mouse secondary antibody (ab128976) for 30min, followed by incubation in avidin-biotin complex for 30min. Finally DAB (ab64238) was used as chromogenic substrate for the detection of Ab binding. Stained sections were counter stained with Mayer's haematoxylin, and dehydrated within ascending grades of alcohol and cleared with two changes of xylene, mounted with cover slip based on DPX mountant, (all reagents from Abcam). Kidney sections were examined under microscope for brown immunoreactivity colour and photos at 400× magnification.

Formalin-fixed kidney sections (5μm) were stained with haematoxylin and eosin to distinguish cell nuclei and digital images of glomeruli were recorded at 400× magnification using a light microscope. Glomerular tuft areas were measured by microscopy computer system (Motic-2000), while, glomerular cellularity was determined by counting the number of nuclei in 20 hilar glomerular tuft cross-sections per animal.

For determining pathological score haematoxylin eosin stained preparations were evaluated under light microscope. Dilated tubules, loss of brush border, tubular casts, leukocytic infiltration and tubular degeneration in the cortical area were scored as described by Biswas et al.21 The scoring system used is described as follows. Kidneys showing no tubular injury were marked 0. While, kidneys exhibiting mild tubular injury ≤10% were given a score of 1. Similarly, kidneys showing mild (10–25%), moderate (26–50%), extensive (=51–75%) and severe (≥75%) tubular injuries were assigned a score of 2, 3, 4 and 5, respectively. Tubular cast scored as 0=negative and 1=positive.

Kidney sections stained with Mason's Trichrome, Gomori silver technique, Caspase 3 in glomeruli and tubules, Kim-1 in glomeruli and tubules, and malondialdehyde gene-expressions by ABC method were quantitatively scored as −=none, +=little, ++=mild and +++=intense.

Statistical evaluation was carried out by using one-way ANOVA test and SPSS (16.0 software), all values were expressed as mean±SD. Values of p<0.05 were accepted as significant.

Kidney index showed insignificant difference between control and gentamicin experimental groups (Table 1). Blood urea nitrogen (BUN) levels increased significantly (p<0.05) in gentamicin (Gm) and gentamicin with propolis (GmP) groups compared to control group. The Kidney index for Control, Gm and GmP group was 22, 41 and 38, respectively (Table 1). It is important to note that there was an insignificant decrease of Kidney index in GmP group (38) compared to Gm group (41). Creatinine levels showed insignificant difference in gentamicin experimental groups compared to control group (Table 1).

Control kidney sections showed normal tubules without dilatation and proximal tubules appeared filled because of the long microvilli of the brush border and aggregates of small plasma proteins bound to this structure, by contrast lumens of distal tubules appeared empty (Fig. 7a). Sections of Gm mice kidney showed mild dilatation with a pathological score of 2 with empty lumens of proximal tubules score (3), moderate loss of pathological score (Fig. 7b). Whereas, sections of GmP mice scored 1, with mild injuries, dilatation and loss of brush borders (Table 3, Fig. 7c).

Fig. 7.

Tubular analysis of control (a), Gm (b), GmP (c) and treated mice.

(0.47MB).

Control sections show (score 0) no leucocytic infiltration, tubular degeneration and tubular cast (Fig. 8a). While sections of Gm mice kidney show severe leucocytic infiltration (score 5, Fig. 8b), extensive tubular degeneration (score 4, Fig. 8c) and presence of tubular cast (Score 1, Fig. 8d, Table 3). Whereas, sections of GmP scored show mild leucocytic infiltration (Score 1), and tubular degeneration but do not show tubular cast (score 0, Fig. 8e).

Fig. 8.

Leucocytic infiltration and tubular degeneration in control (a), gentamicin administered (Gm group; b, c) and GmP mice group.

(0.61MB).

Immunohistochemical analysis of control mice shows no immunoreactivity in control sections (−) for caspase 3 (Fig. 9a1). Whereas, mild (++) immunoprecipitates were seen in tubules kidney of Gm mice (Fig. 9a2). In GmP mice group however, there was a significant decrease in the intensity of immunoprecipitation (+) (Table 4, Fig. 9a3).

Fig. 9.

Immunohistochemical staining of, caspase 3 in control (a1), Gm (a2) and GmP (a3), Kim-1 control (b1), Gm (b2) and GmP (b3) and Malondialdehyde in control (c1), Gm (c2) and GmP (c3) group of mice.

(0.88MB).

Kim-1 gene-expression also shows no immunoreactivity in control sections (−) (Fig. 9b1). Whereas, intense (+++) immunoprecipitation was observed in tubules of Gm mice kidney (Fig. 9b2). Moreover, in GmP mice (little, + Table 4) the intensity of Kim-1 gene immunoprecipitation was very low (Fig. 9b3). Kidney sections stained for Malondialdehyde (oxidative stress Marker) gene-expression showed no immunoreactivity in control sections (−) (Fig. 9c1). Whereas, intense (+++) brownish immunoprecipitates were seen in tubules of Gm mice kidney (Fig. 9c2), and very low intensity of (+, Table 4) immunoprecipitates was found in the tubules of Gmp mice kidney (Fig. 9c3).