Background
The degree of oxidative stress and the severity of subsequent tissue injury may depend on an imbalance between the excessive production of reactive oxygen species and the antioxidant defense. The antioxidants include the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), which detoxify reactive oxygen species. Catalase (E.C.1.11.1.6) is a major enzyme that catalyzes the decomposition of hydrogen peroxide (H
2O
2) and plays a role in cellular antioxidant defense mechanisms [
1]. The main reaction of catalase is the catalytic reaction (2H
2O
2 → O
2+ 2H
2O), which is essential for the removal of excessive H
2O
2 and for regulating the H
2O
2 concentration [
2]. Catalase limits the accumulation of H
2O
2 generated by various oxidases in tissue, and serves as a substrate for the Fenton reaction to produce the highly injurious hydroxyl radicals. Genetic defects of catalase were first documented by Takahara [
3] in Japanese patients who exhibited a deficiency of blood catalase enzyme activity (acatalasemia) [
4,
5]. Subsequently, an acatalasemic mouse strain (Cs
b) was established by Feinstein, Suter, and Jaroslow [
6] from the progeny of x-ray-irradiated mice.
Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in both children and adults [
7,
8]. The clinicopathological syndrome may be classified as primary, secondary or familial. The primary defect in FSGS lies in the filtration barrier of the glomeruli. Disruption of the filtration barrier results in the loss of permselectivity, and macromolecules such as albumin are allowed to enter the urine. Chen et al. [
9] reported that BALB/c mice were susceptible to renal toxicity arising from the administration of the anthracycline antibiotic, adriamycin (ADR), with selective injury to podocytes resulting in severe proteinuria and progressive renal failure [
9,
10]. This was described as the first experimental model of FSGS in mice. The activities of antioxidant enzymes including catalase, GPX and Mn-SOD and the glutathione concentration in renal cortex were decreased by ADR nephropathy in BALB/c mice [
11]. The level of nitric oxide in kidney homogenates [
12], and the urinary levels of nitrite/nitrate [
10] were also increased in the ADR nephropathy model. The administration of the soluble receptor for advanced glycation endproducts (AGEs) suppressed AGE generation and reactive oxygen species in the ADR nephropathy mice [
13].
In the present study, we hypothesized that a defect in the antioxidant system in the form of catalase deficiency would enhance proteinuria, glomerular sclerosis, and eventually lead to the loss of renal function. This hypothesis was tested using the ADR nephropathy model, which is a well-established model of progressive FSGS, in acatalasemic mice.
Discussion
In the present study, acatalasemic mouse strains deficient in catalase activity were used as an animal model. These mice were found to be more susceptible to functional and morphological alterations in the kidneys induced by adriamycin than wild-type mice. The level of albuminuria and glomerulosclerosis in the acatalasemic mice after adriamycin injection was significantly higher than that in the wild-type mice. The renal catalase activity in these mice remained low, without compensatory upregulation of GPX or SOD. Collectively, these data suggest that the increased ROS, particularly the hydroxyl radical, resulting from the reduction of catalase activity, may be involved in the acceleration of glomerulosclerosis found under acatalasemic disease conditions.
Although some rat strains show complete susceptibility to ADR, most mouse strains do not. Zheng et al. showed that AKR/J, C3H/HeJ, CBA/J, C57BL/10J, LP/J, SWR/J, SJL/J, and 129S6/SvEvTac mice were resistant to ADR nephropathy, whereas 129S1/SvImJ and BALB/cByJ mice were susceptible [
26,
27]. They also showed that the susceptible allele for the adriamycin was present in the DOXNPH locus [
28]. We did not confirm whether this allele is present in the C3H/AnL mouse strain. Instead, we performed a mutation analysis of the
TLR4 gene, because it has been thought that TLR4 is involved in progressive renal fibrosis [
29]. TLR4 is considered to be the critical component of the LPS receptor complex. In 1998, a point mutation in the
TLR4 gene was found to be the molecular basis of the LPS hyporesponsiveness in C3H/HeJ mice [
23]. The C3H/AnL mice used in the present study did not have this mutation. Therefore, ADR induced mild renal fibrosis in both groups, although the degree of interstitial fibrosis was only significant in the acatalasemic mice.
ADR induced severe albuminuria, thus leading to values ranging from 23 to 226 mg/mgCr in BALB/cj mice and a value of 0.04 mg/gCr in B6/D2 mice [
30]. In our study, ADR induced relatively mild albuminuria, with 0.6 mg/mgCr in wild-type and 1.5 mg/mgCr in acatalasemic mice. The C3H/AnL mouse strain is considered to have mild sensitivity to ADR. A single dose of ADR (9.5 mg/kg BW) brought about 40% segmental glomerulosclerosis in the BALB/c mouse strain in a previous study [
31]. The wild-type mice which we used in this study had only 15% segmental glomerulosclerosis at week 8 after administration, while the acatalasemic mice had 20% glomerulosclerosis at week 8. This indicates that when the catalase activity is decreased, the resistance to ADR is diminished, and segmental glomerulosclerosis is induced. The number of foot processes of the podocytes did not differ between the mouse groups, indicating that the foot process effacement of the podocytes was affected to a similar degree in both mouse groups. The discrepancy between the level of albuminuria and the degree of foot process effacement may be due to the fact that this evaluation procedure compares only the surviving podocytes in the unsclerosed glomerulus.
Noiri et al. previously investigated the percentage of cortical interstitial volume in the ADR model [
19], and Turnberg et al. evaluated tubulointerstitial injury, including cast formation [
32]. We evaluated the presence of tubulointerstitial injury in similar analyses, and found significant tubulointerstitial injury only in the acatalasemic mice. The fact that acatalasemia exacerbates pulmonary fibrosis in bleomycin-induced lung injury [
33] and chlorhexidine gluconate-induced peritoneal fibrosis [
34] was already demonstrated. In this study, we showed that acatalasemia exacerbates renal fibrosis in a murine FSGS model.
The overexpression of catalase prevented albuminuria and interstitial fibrosis in the angiotensinogen transgenic mice [
35]. In our study, the deficiency of catalase accelerated the albuminuria and glomerular sclerosis in the ADR nephropathy model. These data suggest that reactive oxygen species may contribute to progressive renal injury. Injac et al. reported that the activity of catalase, which detoxifies hydrogen peroxide to H
2O, was increased after ADR administration in rats [
36]. We measured the catalase activity in the whole kidneys of wild-type mice, and found that its level increased after ADR administration. Therefore, the differences in the albuminuria and the degree of glomerulosclerosis between wild-type and acatalasemic mice may be due to the significant difference of catalase activity in kidneys in these mice and the inability of the acatalasemic mice to increase catalase activity under oxidative stress.
Sheerin et al. reported that ADR-injected mice that died in the complement protein C3+/+ group at 6 weeks had a high serum urea level prior to death [
37]. In the current study, all mice survived for at least 4 weeks after the injection of 15 mg/kg BW ADR; however 44.7% of the acatalasemic mice and 40.8% of the wild-type mice died 8 weeks after the administration of ADR. These mortality rates were not significantly different between the two groups (Additional file
1: Figure S1
B). Since the incidence of albuminuria was analyzed in all of the mice that had survived at 8 weeks, but histological studies were not performed in more than half of the mice that survived at 8 weeks, bias might have been introduced into the analysis. We did not take blood samples or evaluate the serum urea levels in ADR-treated mice that died just before the end of the 8 weeks in this study. In addition, the administration of ADR can cause cardiotoxicity [
15] and gastrointestinal toxicity in mice [
38], however we could not confirm these toxicities in our experiments.
Acatalasemia is a rare human disease [
3,
39]. It is unknown whether albuminuria and glomerulosclerosis are related to low catalase activity in humans, although the total antioxidant capacity is correlated with albuminemia, and inversely correlated with proteinuria and anti-DNA antibodies in subjects with lupus nephritis [
40]. In addition, apoptosis, which is thought to be related to oxidative stress, correlated with the immunoserological activities of lupus nephritis [
41] and idiopathic early FSGS [
42]. However, the mechanism(s) by which oxidative stress influences human renal disease is largely unknown. Further studies are needed to elucidate whether and how a low catalase activity in humans influences either albuminuria or glomerulosclerosis associated with kidney diseases.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KT designed the study, carried out the experiments, undertook the data analysis and wrote the manuscript. HS and HM conceived of the study, and participated in its design and coordination, and helped to draft the manuscript. TI, HM, YK and MK participated in experimental sampling. SK and YM undertook the data analyses. NM cooperated with the enzyme activity measurement. DW and KO helped with the animal experiments. All authors read and approved the final manuscript.