Acatalasemic mice are mildly susceptible to adriamycin nephropathy and exhibit increased albuminuria and glomerulosclerosis

Male wild-type mice (C3H/AnLCs^aCs^a) and male homozygous acatalasemic mutant mice (C3H/AnLCs^bCs^b) were used at the age of 8 - 10 weeks. Animals were housed in cages and fed standard chow and water ad libitum. ADR (10, 15 and 20 mg/kg BW) dissolved in saline was intravenously administered to both acatalasemic mutant mice and control wild-type mice [14]. In the C3H/AnL strain, the dose of 10 mg/kg BW did not induce significant albuminuria or increase mortality (Additional file 1: Figure S1A). The 20 mg/kg BW dose resulted in a rapid increase in mortality in both groups of mice (Additional file 1: Figure S1C). The ADR-induced cardiotoxicity might have led to the high mortality resulting from the higher dose of ADR [15]. A total of 55.3% of the acatalasemic mice and 59.2% of the wild-type mice survived 8 weeks after the injection of 15 mg/kg BW ADR (Additional file 1: Figure S1B) with a substantial amount of albuminuria. Therefore, 15 mg/kg BW ADR was considered to be a suitable concentration to investigate the effects of a functional catalase deficiency in the ADR nephropathy model. In the control mice, the same volume of saline was injected intravenously.

Mice were divided into subgroups (n = 6-15/group). Their body weight was measured at 0, 4 and 8 weeks. The mice were sacrificed at 0, 4 and 8 weeks after ADR administration, then their kidneys and hearts were harvested, washed with saline, blotted dry on gauze, and weighed as described previously [16, 17]. The whole kidney weight and heart weight were expressed as a percentage of the body weight determined at the time the mice were sacrificed. Twenty-four hour urine samples were collected in metabolic cages every 4 weeks. Immediately before death, blood samples were drawn. The serum creatinine, blood urea nitrogen (BUN), and urinary albumin excretion (UAE) levels were measured, and the creatinine clearance (Ccr) was calculated as described previously [16]. The experimental protocol was approved by the Ethics Review Committees for Animal Experimentation of Okayama University Graduate School (OKU-2009226).

Chemicals and reagents of analytical grade were purchased from Sigma Co. Ltd. (St. Louis, Missouri) or Wako Pure Chemical Ind. (Osaka, Japan) unless stated otherwise. The mouse monoclonal antibody to 4-hydroxy-2-nonenal (4-HNE) was obtained from Nof Life Science (Tokyo, Japan). The N-histofine MOUSESTAIN KIT was obtained from NICHIREI BIOSCIENCES INC. (Tokyo, Japan). The catalase assay kit was obtained from Cayman (Ann Arbor, Michigan).

The kidneys were removed, fixed in 10% buffered formalin, and embedded in paraffin. Paraffin sections (3-μm thick) were stained with periodic acid-Schiff (PAS) and Masson's trichrome stain. Each tissue section was evaluated under an Olympus light microscope (Olympus, Tokyo, Japan) with a high-resolution digital camera system (Penguin 600CL; Pixera Co., Los Gatos, CA). The area of glomeruli was measured using a Microanalyzer software program (version 1.1, Japan Poladigital Co., Tokyo, Japan) [18]. Glomerulosclerosis was quantified using the percentage increase in the relative mesangial matrix area (the PAS-positive area within the glomerulus divided by the glomerular capillary area; high magnification). All mean values were calculated from 10 glomeruli. Electron microscopy was performed for the mouse kidney specimens as described previously [16, 17]. A quantitative analysis was performed to count the number of podocyte foot processes per 10 μm of glomerular basement membrane in each glomerulus by electron microscopy. The mean number of podocyte foot processes was defined as the effacement score. The area of interstitial fibrosis in the cortex was evaluated with Masson's trichrome as described previously [19], with some modifications. Under low magnification, the number of casts in five randomly selected non-overlapping fields from the cortical region was counted and averaged as the cast score.

To detect lipid oxidation products, paraffin sections (3-μm thick) were stained using an N-histofine MOUSESTAIN KIT as described previously [20]. A mouse monoclonal antibody to 4-HNE was used as the primary antibody (1:100 dilution). Under low magnification, five randomly selected non-overlapping fields from the cortical region were analyzed. The 4-HNE positive areas that were stained in brown were picked up on digital images, and the percentage of the 4-HNE positive area relative to the whole area of the field was calculated (% area).

Kidney samples were stored in a -80°C freezer until being assayed. The catalase activity in each kidney was determined by an ELISA using a catalase assay kit [21]. All procedures were performed according to the manufacturer's instructions.

The detection of the Tlr4 mutation was performed using nested-PCR as described previously [22]. The nested PCR for detection of the missense mutation, a C to A transversion (Pro712His), which was reported in the C3H/HeJ mouse strain [23], was performed using the primers shown in Additional file 2: Table S1. The primers TLR4-ex3-F and TLR4-ex3-R were used for direct sequencing of DNA extracted from the mouse kidneys.

The data were shown as the means ± SE. The normal distribution and homogeneity of variance were checked, and logarithmic transformations were made for the variables if needed. Multiple comparisons between groups were made by Scheffe's test or the Steel-Dwass test. A Kaplan-Meier analysis and the Log-Rank statistic were used to explore the effects of ADR in both groups of mice. The statistical analysis was performed using the Excel add-in software Statcel 2 program (OMS, Tokyo, Japan) or the JMP 9 software program (SAS Institute Inc., Cary, North Carolina). P < 0.05 denoted the presence of a statistically significant difference.

The ADR nephropathy model has been extensively studied in animals of the BALB/c background. Therefore, we first tried to characterize this model in our mouse strains. The body weight, relative kidney weight and relative heart weight were similar between the groups at the start of the experiment (Table 1). Both groups of mice lost a similar amount of body weight by 8 weeks after ADR administration. However, the relative kidney weight in acatalasemic mice was lower at 8 weeks than that in wild-type mice after ADR administration. The relative heart weights were similar for both groups during the experimental period.

To evaluate the effect of acatalasemia on ADR-induced alterations of biological functions, we measured the parameters of renal function (Table 1) and UAE (Figure 1). The basal levels of BUN and Ccr were not significantly different between the wild-type and acatalasemic mice. In both groups, the renal function did not significantly change during the experimental period. However, UAE was elevated significantly after ADR administration at weeks 4 and 8 in both groups. The UAE in the acatalasemic mice was higher than that in wild-type mice after ADR administration at week 4.

At the start of the experiment, no histological abnormalities of the glomeruli were observed at the light microscopic level in either group (Figure 2A and 2B). The glomeruli in the acatalasemic mice were significantly hypertrophied after ADR administration at week 4 (Figure 2D and 2G). In the acatalasemic mice, glomerulosclerosis developed earlier than in wild-type mice, and it was significantly increased at week 4 compared with that in wild-type mice after ADR administration (Figure 2C through F and 2H). A decrease in the effacement score of the podocyte foot processes was observed in glomeruli of both groups of mice (Additional file 3: Figure S2A through F), however, there were no significant changes in the effacement scores in either group at week 4 and 8 after ADR administration (Additional file 3: Figure S2G). Tubulointerstitial fibrosis developed significantly in the kidneys of acatalasemic mice group (Figure 3B, D, F and 3G) at 4 and 8 weeks after ADR administration, with significant cast formation noted at week 4 (Figure 3H). The tubulointerstitial changes in the kidneys of wild-type mice after ADR administration were not statistically significant compared to that of acatalasemic mice (Figure 3A, C, E, G and 3H).

Modification of proteins and lipids by oxidative stress is believed to play a central role in a variety of biological activities, such as apoptosis and extracellular matrix expansion [18, 24, 25]. The major content of the cell membrane is lipids, and thus, lipid peroxidation may cause renal injury. We next examined whether acatalasemia influenced the lipid peroxidation products in the tubulointerstitial compartment of the kidneys in ADR nephropathy. At the start of the experiment, there was almost no accumulation of 4-HNE in the kidneys of either group (Figure 4A and 4B). There was an increase in the 4-HNE antibody labeling of various patches in the tubules of kidneys after ADR administration in both groups (Figure 4C through 4F). The degree of 4-HNE accumulation in the cortex of the kidneys in acatalasemic mice was significantly higher than that in wild-type mice 8 weeks after ADR administration (Figure 4G).

The maintenance of tissue homeostasis requires an appropriate balance between oxidants and antioxidants. Two major antioxidant enzymes, catalase and GPX, are physiologically involved in the detoxification of H₂O₂, and thus protect tissues from oxidant-mediated injury. Therefore, we next examined the catalase activity in acatalasemic ADR kidneys. Although the renal catalase activity of the wild-type mice was increased significantly at week 4 after ADR administration, that from the acatalasemic mice was decreased compared with wild-type mice at the start of the experiment, and remained low during the entire experimental period (Figure 5). Since high catalase levels are found in erythrocytes, we compared the catalase activity between saline-perfused and unperfused kidneys after the removal of residual blood. The catalase activity of the perfused kidney was 6.0 ± 0.2% lower than that of the unperfused kidney [17]. Since the catalase activity was enhanced in wild-type mice 4 weeks after ADR administration and was thought to contribute to reactive oxygen elimination, we investigated the levels of other renal antioxidant enzymes, such as GPX and SOD, in wild-type or acatalasemic mice 4 weeks after ADR administration. The activity levels of GPX and SOD did not show any remarkable changes in either of the groups of mice (data not shown). Since SOD catalyzes the conversion of the superoxide anion radical to H₂O₂ and O₂, it has been suggested that the level of production of ROS mediated by SOD may be similar in the ADR-nephropathy kidneys in both groups.

To examine the background of the mouse strains, we performed a mutation analysis of Tlr4 exon 3. Tlr4 did not show a C to A transversion due to a missense mutation in the C3H/AnL mouse strain, which was reported to be present in the C3H/HeJ strain (Additional file 4: Figure S3) [23].