Twenty-five male Golden-Syrian Hamsters (Mesocricetus auratus), weighing approximately 140 g each, were used divided into 3 groups. All of the subjects were housed in a room with a 12/12-h light/dark cycle, and an ambient temperature between 22 °C and 25 °C under specific pathogen-free conditions. The experiment took place in the Bioterium located at the Centro Universitario de Ciencias de la Salud (CUCS), from the Universidad de Guadalajara. The first group of hamsters (10 animals) was fed a standard diet (SD), consisting (as a percentage of total kcal) of 12% fat, 60% carbohydrates, and 28% protein. The second group (10 animals) was fed a hypercaloric diet (HCD), which consisted of the SD enriched with 1% cholesterol, 10% coconut oil, and 15% sugar. All animals (SD and HCD groups) were fed for one month. All of the subjects were previously induced to diabetes by an intraperitoneal injection with streptozotocin (STZ, Sigma-Aldrich) in sodium-citrate buffer (0.2M, pH 4.5), once a day for 3 consecutive days at a dose of 50, 40 and 40 mg/kg, respectively (0.3 mg/g approximately). Hamsters had free access to food, water and continued on their original diets for the duration of the study. A group HCD fed (without STZ-treatment) was included as control. After four months, the animals were anesthetized with Ketamine (65 mg/kg) and Xylazine (7 mg/kg) in order to then be sacrificed for biological sample obtaining (Figure 1). All experiments were conducted according to the internationally accepted principles for the care and use of laboratory animals, and the guidelines of the Animal Research Reporting of in vivo Experiments (ARRIVE). It is worth mentioning that the researchers have experience handling laboratory animals and were trained through Internal Committee for the Care and Use of Laboratory Animals (CICUAL) courses and respecting the Official Standard of Technical Specifications for the Production, Care and Use of Laboratory Animals (NOM-062-ZOO-1999).

Figura. 1

The blood glucose levels were monitored using the One-touch Ultra glucose monitoring system (Lifescan Johnson & Johnson Company), and hamsters were considered to be diabetic when three consecutives blood glucose determinations resulted in 200 mg/dL or above. To perform a glucose curve, glucose measurements were taken on days 3, 8, 16, 22, 29, 49, 58 and 73 after the last STZ-administration.

Urine samples were collected twenty-four hours before death from each animal (in order to determine 24-h urinary volumes) by placing the hamsters in a previously cleaned metabolic cage. In addition, approximately 10 mL of blood were obtained from cardiac puncture. Uric acid, total proteins, creatinine, albumin, urea and sodium (Na), potassium (K), and chlorine (Cl) electrolytes in blood and/or urine, were measured following standard methods in an instrument of analysis (Shyncron CX9). The GR was determined following the equation: GFR = (uCr*uV)/(sCr) [13, 14].

Kidneys were quickly removed, set in 10 % phosphate-buffered formalin solution and then embedded in paraffin. Kidneys were cut into semithin sections of 4 - 5 um. The sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome satin (HYCEL SA of CV, México). A single pathologist, unaware of the experimental protocol, analyzed all of the kidney’s slides using a light microscope.

The extent of renal injury was assessed by a histopathologic analysis of the glomerular sclerosis and interstitial alterations (tubular and renal atrophy replaced by fibrous connective tissue), in representative renal tissue of each experimental group. The glomerulosclerosis, renal tubule damage (tubular atrophy), and interstitial fibrosis are cataloged in five groups, between 0 and 4. Glomerulosclerosis, renal tubule damage (tubular atrophy), and interstitial fibrosis are categorized into five groups, from 0 to 4. A score of 0 indicates the absence of damage; a score of 1 represents less than 25 % of minor damage; a score of 2 corresponds to approximately 25 % to less than 50 % of damage, around the median. A score of 3 denotes about 50 % to 75 % of damage (approximately two-thirds of the area), and a score of 4 indicates damage present throughout the entire area (> 75 %) (Vázquez-Méndez et al., 2020).

Masson’s trichrome-stained renal slides from each animal were analyzed, in particular measuring the area, the diameter and the fibrosis of glomeruli. The damage of renal tissue was evaluated in a computational system of image analysis, using an Olympus BX51 microscope equipped with a DP71 camera, and Image-ProPlus 6.3 software from Media Cybernetics. From each group of animals (n = 4), 50 glomeruli were analyzed in 20 random fields (magnification 40X) of tissue sections.

Total collagen was tested by measuring the concentration of hydroxyproline as described by Rojkind et al. (1974). Briefly, renal tissue was hydrolyzed with 6N HCl at 110°C for 24 hours. Samples were incubated with a chloramine-T buffer for 10 m at room temperature. Ehrlich’s reagent was added, and the samples were again incubated for 45 minutes at 65 °C. Absorbance of each sample was measured at 560 nm using a spectrophotometer (Thermo Scientific, Biomate 3, USA). Collagen accumulation was expressed as percent of collagen in each experimental group.

The data are expressed as mean ± SD. Analysis through the t-Student test was used to compare the differences between groups. Values of P < 0.05 were considered significant.

Approximately 95 % of hamsters that receive HCD+STZ, manifested severe hyperglycemia (plasma glucose level of 250-350 mg/dL throughout all of the experiment) as shown in figure 2A. Approximately 30 % of the animals, which exhibited significant weight loss (40 g) and dehydration, as shown in Figure 2B, were humanely euthanized by cervical dislocation, performed by trained personnel in accordance with ethical guidelines to prevent further suffering. Death was confirmed by the absence of reflexes and cardiac activity. The hamsters that received SD+STZ showed only mild hyperglycemia (140-160 mg/dL). The undernourishment and dehydration were not as evident, but said animals still presented moderate weight loss (18 g) when compared to the former group. The remaining hamsters that were only HCD fed did not present hyperglycemia, undernourishment nor dehydration.

Figure 2

The biochemical analysis demonstrated that the serum uric acid increased 28.7 % and 73 % respectively in both animal groups, the SD group, and the HCD that was treated with STZ. In urine, uric acid only decreased (61%) in diabetic animals induced by STZ that were fed with HCD. Urea in serum is normal in all 3 groups, but urine increased significantly (138 %) in diabetic animals induced with STZ plus HCD (in STZ plus SD, the group increase was 27 %). Serum creatinine (Cr) concentrations increased slightly at the end of experiment in both animal groups that were treated with STZ. Moreover, uric Cr significantly decreased (78%) in the experimental group treated with STZ plus HCD, while in the STZ + SD group, it decreased 31%. All of this data indicates a tendency consistent with chronic and progressive renal failure. Total proteins experienced no changes (urine or serum) in the 3 groups. Serum albumin decreased significantly (8%) in the animals which received STZ + HCD and increased (31%) in urine (Table 1). In the electrolytes analysis related to urine, sodium decreased in both STZ-treated groups, while in serum no significant changes were present. In serum, potassium showed an increase of 20 % exclusively in the animals that received STZ + SD. While in urine, the potassium decreased significantly to 66 % in this same group (the other group treated with STZ decreased as well, but in less proportion - 21 %). In serum, chlorine increased in both STZ-treated groups to 10 %. Nevertheless, in urine, these same groups significantly decreased the concentration to 50 % for STZ + SD and 100 % for STZ+HCD.

Tabla 1

	Uric Acid (mg/dL)		Total Proteins (g/dL)		Albumin (mg/dL)		Urea (mg/dL)		Creatinine (mg/dL)
Group	Serum	Urine	Serum	Urine	Serum	Urine	Serum	Urine	Serum	Urine
HCD	1.5 ± 0.61*	10.8±2.42*	5.1 ± 0.59	0.1 ± 0.08	2.2 ± 0.28	156.5 ± 106.4	32.7 ± 3.2	261.2 ± 111	0.45 ± 0.06	143.33 ± 28.8
SD + STZ	1.9 ± 0.54	11.2±4.42*	5.33 ± 0.33	0.1 ± 0.14	2.3 ± 0.10*	152.2 ± 33.19	35 ± 1.2	333.7 ± 169	0.46 ± 0.13	103.26 ± 46.4
HCD + STZ	2.6 ± 0.71 *	4.2 ± 4.8 *	5.2 ± 0.57	0.1 ± 0.10	2.1 ± 0.07 *	205.6 ± 60.6	35.5 ± 4.9	621 ± 313.6 *	0.61 ± 0.36	32.87 ± 22.2 *

Table 1. Biochemical analysis in each experimental group. Table 1A show concentration (mg/dL) of uric acid, total proteins, albumin, urea and creatinine (urine and serum) in each experimental group. Table B, show the Na, K and Cl electrolytes levels (mM/L) in serum and urine in each treatment. The results are presented as means ± standard deviation (SD). Differences are considered statistically significant when p < 0.05. Statistically significant results are marked with an asterisk (*).

Table B

Group	Na (mM/L)		K (mM/L)		Cl (mM/L)
	Serum	Urine	Serum	Urine	Serum	Urine
HCD	132.5 ± 14.7	137.8 ± 50.6	6.4 ± 0.5*	241.6 ± 9.9*	91.8 ± 9.6	114.1 ± 83.9
SD + STZ	145.4 ± 0.94	107 ± 60.6	6.2 ± 0.4*	190.7 ± 56.7*	101.2 ± 1.2	58.24 ± 46.5
HCD + STZ	140.4 ± 7.2	107.5 ± 47.2	7.7 ± 0.6 *	83.2 ± 39.7 *	100.9 ± 2.8 *	0 ± 0

GFR was calculated with respect to renal creatinine clearance, in 24-h urine collection (Figure 3A). A single measurement of serum creatinine was done, as indicated by the Cockcroft-Gault equation. As shown in Figure 3B, the GFR in HCD and in SD plus STZ groups was similar (0.671 and 0.672 mL/min, respectively), but in diabetic animals, HCD fed, the GFR decreased significatively, around 30 % (0.491 mL/min).

Figure 3.

The glomerular tissue damage was determined to traverse histomorphometric changes of glomeruli, characterized by ECM accumulation in the intra- and extra-glomerular space. This alters the circular shape, whose evolution can progress to functional atrophies or Kimmelstiel-Wilson lesion (Islas Andrade and Revilla Monsalve, 2005), and in addition, presents extravasations even in the Bowman’s space. The interstitial matrix was determined by the presence or absence of vacuolization-glycogen drops (Armanni-Ebstein changes), which have been related to hyper-glucose (Islas Andrade and Revilla Monsalve, 2005). The animals of the HCD group presented enlargement of the capillary wall, mesangial-intercellular expansion, decrement of the space in the Bowman’s capsule, tubule interstitial vacuolization, loss of luminal area, initial changes in the presence of ECM proteins, and an increment of inter-tubular space, as shown in Table 2. Animals of the SD-STZ group showed similar characteristics, though more prominent, alongside glomerular atrophy. Notably, animals that received SD plus STZ demonstrated an exacerbation of significantly aggressive features and atrophy with extrusion into Bowman’s space. Furthermore, significant evidence of glomerulosclerosis, tubular atrophy, and interstitial fibrosis was observed after feeding animals with HCD-STZ, as shown in Table 3. At the end of the experiments, the score of glomerulosclerosis, tubular atrophy, and interstitial fibrosis was 1 in the HCD group; glomerulosclerosis (2), tubular atrophy, and interstitial fibrosis (1) in the SD group. This suggests that, by the end of the experiments, renal scarring was moderate to severe in both groups. Nevertheless, in the HCD-STZ group, glomerulosclerosis and interstitial fibrosis (3 and 2 scores, respectively) were exacerbated.

Table 2

CHARACTERISTICS	HCD	SD-STZ	HCD-STZ
Thickening of capillary wall	✓	✓	✓
Intercellular-mesangial expansion	✓	✓	✓
Decrease in space in Bowman's capsule	✓	✓	✓
Tubulo-interstitial vacuolization	✓	✓	✓
Loss of the luminal area	✓	✓	✓
Increment of ECM proteins	✓	✓	✓
Increase in inter-tubular spaces	✓	✓	✓
Glomerular atrophy	✓	✓	✓
Atrophy with extrusion into Bowman's space	✓	✓	✓

Table 4.

Characteristic	HCD	SD-STZ	HCD-STZ
Glomerular sclerosis	1	2	3
Tubular atrophy	1	1	1
Interstitial fibrosis	1	1	2
Total	3	4	6

Renal fibrosis evaluated by a morphometric analysis and hydroxyproline biochemical determinations, demonstrated a significant increase only in the animals that receive HCD+STZ, as much in glomeruli, interstice, and crust (36.3, 75.2 and 70.7 %, respectively) (Figure 4 E, F). The SD+STZ group (Figure 4 C, D) presented a slight increase of glomerular, interstitial, and cortical extracellular matrix (1.7, 12.5 and 11.2 %, respectively) with respect to HCD group (Figure 4 A, B). The progression of collagen deposition in kidney tissue was also confirmed by the measurement of tissue hydroxyproline content, that progressively increased in the HCD+STZ group. Results obtained with histological analysis and hydroxyproline determinations showed structural changes in renal tissue during the development of kidney fibrosis.

Figure. 4