Chemicals

All reagents used in the present study were of analytical grade. Carbon tetrachloride was obtained from sd Fine Chemicals, Bhoisar, Mumbai, India. 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB), L-Glutathione (reduced form), and all current chemicals were bought from Sigma Chemical Co., (St. Louis, MO, USA).

Bacterial strains and growth conditions

Lactococcus lactis subsp.cremoris was isolated in a previous study. Standard cultures were prepared by inoculation of 10 mL M17 broth (Biokar Diagnostics) or MRS (Accumix) with 100 µl of frozen strains stock and incubated for 24 h at 37 °C. Then, 100 µl of the 24 h grown strain was reinoculated into 10 ml MRS broth for 24 h at 37 °C. To adapt bacteria to milk fermentation conditions, strains working cultures were prepared by inoculating (1%) fresh precultures in sterile (100 °C, 20 min) camel milk and incubated (12 h, 37 °C).

Sample preparation

The fresh camel milk sample was collected from the local farm on May 2016 (Sfax, Tunisia). The sample was cooled immediately at 4 °C and then blended to obtain a homogenous sample followed by the pasteurisation process. Fresh milk was heated at 80 °C for 20 min and cooled to 43–45 °C (Moslehishad et al. Reference Moslehishad, Mirdamad, Ehsani, Ezzatpanah and Moosavi-Movahed2013).

Batch culture experiments were performed in the pasteurised fresh milk. Pure and mixed cultures were carried out without any mechanical agitation. After 24 h of fermentation, the samples were centrifuged for 15 min at 15 000 × g and supernatant used in the assays.

Animals and experimental design

This study was carried out on 28 female mice weighing 29 ± 2 g, purchased from the Central Pharmacy of Tunis (SIPHAT). After 15 d adaptation period, the mice were randomly and equally divided into four groups of seven animals each: group1 (control group [C]); Group 2 (CCl₄ group [CCl₄]); Group 3 (Fermented Camel Milk + CCl₄ group [FCM + CCl₄]); Group 4 (Fermented Camel Milk group [FCM]).

Groups 2 and 3 received a single dose of CCl₄ (10 ml/kg in 0·3% olive oil. ip) at 14 d. Mice of the control group (group 1) received an equal volume of olive oil. Groups 3 and 4 were administrated camel milk fermented using lactococcus lactis subsp cremoris by oral gavage (0·4 ml/kg b.w/day) during 15 d before CCl₄ injection. Twenty-four hours after the CCl₄ challenge, all animals were weighed and then sacrificed by cervical decapitation to avoid stress.

The plasma was obtained by centrifugation (4000 rpm g, 15 min, 4 °C) and stored at −20 °C for biochemical assays. The kidneys of each animal were removed, cleaned, weighed and rinsed with a physiological saline solution. Samples were rinsed, homogenised in tris buffer solution (50 mm; pH 7·4) using an Ultra-Turax Homogeniser and centrifuged (5000 × g for 25 min at 4 °C). The supernatant was kept at − 20 °C until analyses. In parallel, portions of kidney were immediately fixed into in 10% buffered formalin solution for histological studies.

Biochemical parameters

The levels of urea, uric acid, and creatinine in plasma were assayed by autoanalyser (Erba xl 200, Mannheim, Germany) using Erba diagnostic kit.

The levels of sodium, potassium, calcium, chloride, and phosphorus in plasma were assayed by autoanalyser (Erba Lyte pro, Mannheim, Germany).

Lactate dehydrogenase (LDH) activities in the kidney and plasma were measured using a commercially available diagnostic kit (Biomaghreb, Tunisia, Ref. 200126).

Oxidative damage assays

The level of lipid peroxidation in kidney was estimated according to the method of (Yagi, Reference Yagi1976). Briefly, an aliquot of kidney extracts supernatant was mixed with 1 ml of 5% trichloroacetic acid and centrifuged at 2500 × g for 10 min. An amount of 1 ml of thiobarbituric acid reagent (0·67%) was added to 500 µl of supernatant and heated at 90 °C for 15 min. The mixture was cooled and the absorbance was recorded at 765 nm. Likewise, protein carbonyl (PCO) content in kidney tissue was measured using the DNPH method of Reznick & Packer (Reference Reznick and Packer1994). Briefly, after precipitation of protein with an equal volume of 1% trichloroacetic acid, the pellet was resuspended in 10 mm/l DNPH plus 2N HCl, or in 2N HCl as a control blank. Next, after the washing procedure with 1 : 1 ethanol-ethylacetate, the final palette was dissolved in 6 m/l guanidine. The carbonyl group was determined from the absorbance at 370 nm. Results were expressed in nm/mg protein. Protein content was evaluated as described by Lowry et al. (Reference Lowry, Rosebrugh and Farr1951) using bovine serum albumin as standard.

Assessment of antioxidant status

Superoxide dismutase (SOD) activity was estimated according to Beyer & Fridovich (Reference Beyer and Fridovich1987).

Catalase activity was assayed by H₂O₂ consumption method (Aebi, Reference Aebi1984). GPx activity was measured according to the method of Flohe & Gunzler (Reference Flohe and Gunzler1984). GSH in plasma was measured by the method of Ellman (Reference Ellman1959) modified by Jollow et al. (Reference Jollow, Mitchell, Zampaglione and Gillette1974).

The determination of vitamin C content in the kidney tissue was performed according to Jacques-Silva et al. (Reference Jacques-Silva, Nogueira, Broch, Flores and Rocha2001).

Histopathological examination

Kidneys removed from the control and tested mice were cleaned and fixed in Bouin solution. Then they were embedded in paraffin and stained with haematoxylin–eosin for histopathological studies. All sections were evaluated for the degree of tubular and glomerular injury and necrosis. Six slices from each kidney were examined and assigned for severity of changes using scores on a scale of none (−), mild (+), moderate (++), and severe (+++).

Statistical analysis

Statistical analyses were performed using one-way ANOVA followed by Fisher's post-hoc test (PLSD) for comparison between groups. Data are expressed as mean ± se. Differences were considered significant if P < 0·05.