Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513 Japan
Correspondence and offprint requests to: Yuichi Kikuchi, MD, Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513 Japan. Email: grd1615{at}ndmc.ac.jp
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Abstract |
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Methods. Male Otsuka LongEvans Tokushima Fatty (OLETF) rats, a non-insulin-dependent diabetic model, and LongEvans Tokushima Otsuka (LETO) rats, a non-diabetic control, were studied at 35 weeks of age. OLETF rats were randomized to receive mizoribine (5 or 10 mg/kg) or normal saline for 8 weeks. Histological changes such as glomerulosclerosis and interstitial fibrosis and the number of ED1- and CD5-positive cells in the kidney were assessed. By using reverse transcriptionpolymerase chain reaction (RTPCR) and immunohistochemistry, monocyte chemoattractant protein-1 (MCP-1), osteopontin (OPN) and transforming growth factor (TGF)-ß1 expression in the kidney was also analysed.
Results. Urinary albumin excretion in OLETF rats increased compared with that in LETO rats. Administration of mizoribine suppressed urinary albumin excretion. Development of glomerulosclerosis, interstitial fibrosis and macrophage infiltration in the kidney was also inhibited by treatment with mizoribine. The expression of MCP-1, OPN and TGF-ß1 mRNA in untreated OLETF rats was significantly increased compared with that in LETO rats. By immunohistochemistry, increased expression of MCP-1, OPN and TGF-ß1 was found in the tubules and glomeruli of untreated OLETF rats. This expression was significantly suppressed by treatment with mizoribine.
Conclusions. Mizoribine inhibited renal macrophage accumulation and prevented the progression of glomerulosclerosis and interstitial fibrosis in non-insulin-dependent diabetic kidneys. In addition to standard treatments, anti-inflammatory agents may be useful for management of non-insulin-dependent diabetic nephropathy.
Keywords: anti-inflammatory agent; diabetic nephropathy; monocyte chemotactic protein-1; osteopontin; transforming growth factor-ß1
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Introduction |
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Mizoribine is an imidazole nucleoside isolated from Eupenicillium brefeldianum. Mizoribine inhibits the conversion of inosine 5'-nucleotide to guanosine 5'-nucleotide in the purine nucleotide biosynthetic pathway, and shows immunosuppressive effects similar to MMF [10]. The efficacy of this agent in patients having not only renal transplantation, but also rheumatoid arthritis, lupus nephritis and primary nephrotic syndrome has been demonstrated in Japan. Moreover, the incidence of adverse effects with this drug, which include myelosuppression, hepatotoxicity and nephrotoxicity, are rare compared with other immunosuppressive agents [11].
Based on these findings, we examined whether mizoribine suppressed macrophage infiltration in the kidneys and reduced renal damage in a spontaneous non-insulin-dependent diabetic model, the Otsuka LongEvans Tokushima Fatty (OLETF) rat. These animals are obese and have hyperglycaemia, hyperinsulinaemia, hyperlipidaemia and hypertension [6,8,12]. We focused on whether administration of mizoribine exerted renoprotective effects on OLETF rats, which already showed overt diabetic nephropathy. We also examined the effect of mizoribine on the expression of MCP-1, osteopontin and TGF-ß1, which is closely related to renal macrophage infiltration and the progression of renal injury.
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Materials and methods |
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Experimental protocol
We used pathogen-free male OLETF at 35 weeks of age and age-matched LETO, non-diabetic control rats. Rats were given free access to tap water and standard rat chow. Animal care followed the guidelines of the National Defense Medical College for the care and use of laboratory animals in research. Mizoribine was dissolved in normal saline just before injection. Fourteen OLETF rats were given mizoribine (5 or 10 mg/kg, seven in each) intraperitoneally once a day for 8 weeks. Seven OLETF rats were given similar injections with normal saline for the same time period as controls. At the end of the study, systolic blood pressure was measured by the tail-cuff method. Total blood glucose, obtained from the tail vein, was measured by the glucose oxidase method. Following anaesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg), the kidneys were removed. Blood was collected from the abdominal aorta and urine was collected by bladder puncture (spot urine). Serum creatinine, total cholesterol, triglyceride, alanine aminotransferase and urinary creatinine were measured by standard methods using an automatic analyser (Hitachi 7170, Hitachi, Tokyo, Japan). Urinary albumin was measured by using a turbidimetric immunoassay kit (Shibayagi, Gunma, Japan). One kidney was rapidly frozen in liquid nitrogen, and the other kidney was fixed in 10% formalin and embedded in paraffin.
Histological study
All formalin-fixed kidney sections (3 µm) were stained with periodic acidSchiff (PAS) and Masson trichrome. Fifty full-sized glomeruli for each specimen were assessed on PAS-stained sections under a high power field (x400) and the levels of glomerulosclerosis in each glomerulus were scored semi-quantitatively as follows: 0 = no sclerosis; 1 = sclerosis in <25% of glomeruli; 2 = sclerosis in 2550% of glomeruli; 3 = sclerosis in >50% of glomeruli. To evaluate interstitial fibrosis, 20 fields for each section were assessed on Masson trichrome-stained sections (x200). Semi-quantitative analysis in each field was assessed as follows: 0 = no fibrosis; 1 = fibrosis in <10% of areas; 2 = fibrosis in 1025% of areas; 3 = fibrosis in 2550% of areas; 4 = fibrosis in >50% of areas. Averages of glomerulosclerosis and interstitial fibrosis scores were calculated from total evaluated glomeruli or interstitial lesions in each section. These microscopic evaluations were performed by histologists that were blinded to the experimental groups.
Immunohistochemistry
Serial sections (3 µm thick) were used to detect MCP-1, osteopontin, TGF-ß1, ED1 and CD5. Deparaffinized sections were incubated with 3% H2O2 for 10 min to block endogenous peroxidase activity. After blocking with 7% bovine serum albumin or 10% skim milk (for osteopontin) in phosphate-buffered saline (PBS), each section was incubated with a primary antibody for MCP-1, osteopontin, TGF-ß1, ED1 and CD5 (0.5, 1, 12.5, 5 and 10 µg/ml, respectively) for 4560 min at 37°C. The staining procedure for ED1 and CD5 required treatment with proteinase K for 7 min for optimal antigen retrieval. Thereafter, the sections were incubated with a DAKO LSAB system link antibody for 10 min, followed by incubation with peroxidase-conjugated streptavidin for 10 min at room temperature. After washing with PBS, the sections were stained with a 3,3'-diaminobenzidine (DAB) solution and then counterstained with haematoxylin. More than 50 glomeruli and 20 non-overlapping interstitial areas from each section were assessed under high power magnification (x400), and the numbers of ED1- and CD5-positive cells in each glomeruli and interstitial area were counted and averaged in each group.
RNA extraction and RTPCR
Total RNA was extracted from the renal cortex using a phenolchloroform extraction method. RNA was quantified by absorbance at 260 nm. A 5 µg aliquot of total RNA was incubated in a reverse transcription mixture (total volume 20 µl), containing 0.5 µg of oligo (dT), 0.5 mmol/l dNTP mix and 200 U of reverse transcriptase (SuperScript II), at 42°C for 50 min. Reverse transcription was stopped by heating the samples at 72°C for 15 min, after which samples were stored at 4°C until polymerase chain reaction (PCR) was performed.
The MCP-1 sense primer was defined by bases 5170 (5'-GCCAGATCTCTCTTCCTCCA-3'), and the antisense primer by bases 463482 (5'-GAGGTGGTTGTGGAAAAGAG-3') (GenBank accession no. NM031530). The cDNA PCR amplification product was predicted to be 432 bp in length. The osteopontin sense primer was defined by bases 160179 (5'-AGAGGAGAAGGCGCATTACA-3'), and the antisense primer by bases 638657 (5'-GCAACTGGGATGACCTTGAT-3') (GenBank accession no. M14656). The cDNA PCR amplification product was predicted to be 498 bp in length. The TGF-ß1 sense primer was defined by bases 641660 (5'-TACAGGGCTTTCGCTTCAGT-3'), and the antisense primer by bases 10151034 (5'-TGGTTGTAGAGGGCAAGGAC-3') (GenBank accession no. AY55025). The cDNA PCR amplification product was predicted to be 394 bp in length. A 2 µl aliquot of cDNA samples was amplified in a 50 µl PCR mixture. Each primer was used at a concentration of 0.15 µmol/l. The annealing temperature for MCP-1 was 62.0°C and that for osteopontin and TGF-ß1 was 61.0°C. The PCR for MCP-1 and TGF-ß1 was carried out for 28 cycles and that for osteopontin for 24 cycles.
To quantify the expression of these mRNAs, competitive PCR was performed. The mRNA levels were normalized to the levels of ß-actin mRNA. The competitors were synthesized with a competitive DNA construction kit (Takara, Shiga, Japan). The sizes of the competitors for MCP-1, osteopontin and TGF-ß1 were 340, 390 and 290 bp, respectively. After PCR, 10 µl of PCR product was transferred into another tube and 2 µl of 6x loading buffer was added. Samples were electrophoresed in a 1.5% agarose gel in TAE. The PCR products were visualized by ethidium bromide staining. The expression of MCP-1, osteopontin and TGF-ß1 mRNA was quantified by comparison with that of competitors using a densitometer (ATTO, Tokyo, Japan).
Statistical analysis
Results are expressed as means±SEM. Statistical analyses were performed using analysis of variance (ANOVA) followed by Fisher's multiple comparisons. Correlations between glomerulosclerosis, interstitial fibrosis, MCP-1, osteopontin or TGF-ß1 mRNA and the number of ED1-positive cells were assessed by Spearman's correlation test. A P-value of <0.05 was considered significant.
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Results |
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Histological findings
Glomerular lesions in OLETF rats were characterized by hyalinosis, thickening of the basement membrane, mesangial expansion and sclerotic lesions (Figure 1B). The glomerulosclerosis score in untreated OLETF rats increased significantly compared with that in LETO rats (1.05±0.06 vs 0.14±0.04, P<0.001; Figure 2A). Treatment with 10 mg/kg of mizoribine significantly suppressed glomerulosclerosis in OLETF rats (0.84±0.01, P<0.01 vs untreated OLETF; Figures 1C and 2A). Interstitial fibrosis in OLETF rats was focal and mild during the study period. However, the interstitial fibrosis score was greater in untreated OLETF rats than in LETO rats (0.49±0.01 vs 0.13±0.02, P<0.001; Figure 2B). Interstitial fibrotic lesions in OLETF rats were significantly improved by treatment with 10 mg/kg of mizoribine (0.49±0.01 vs 0.36±0.03, P<0.01). Our preliminary study showed that OLETF rats at 35 weeks of age (n = 5) already had a 0.71±0.04 glomerulosclerosis score and a 0.31±0.01 interstitial fibrosis score. Glomerulosclerosis and interstitial fibrosis scores in untreated OLETF rats were significantly increased compared with those in OLETF rats at 35 weeks of age, whereas these parameters in OLETF rats treated with 10 mg/kg of mizoribine were not different from those at 35 weeks.
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MCP-1, osteopontin and TGF-ß1 expression
MCP-1 mRNA expression in the renal cortex was significantly increased in untreated OLETF rats compared with LETO rats (181%, P<0.01). Although treatment with 5 mg/kg of mizoribine in OLETF rats reduced MCP-1 mRNA expression by 34%, this reduction was not significant. Overexpression of MCP-1 mRNA in OLETF rats was significantly suppressed by treatment with 10 mg/kg of mizoribine by 59% (Figure 4A and B). Osteopontin mRNA expression in untreated OLETF rats was also increased compared with that in LETO rats (173%, P<0.01). Treatment with 10 mg/kg of mizoribine in OLETF rats significantly reduced osteopontin mRNA expression by 31% (Figure 4A and C). TGF-ß1 mRNA expression in untreated OLETF rats was mild, but was significantly increased compared with that of LETO rats (77%, P<0.01). Administration of mizoribine, even though at a dose of 5 mg/kg, markedly suppressed TGF-ß1 mRNA expression, and there was no significant difference in TGF-ß1 mRNA expression between LETO rats and mizoribine-treated OLETF rats. There were significant correlations between the number of ED1-positive cells in the interstitium, but not in glomeruli, and mRNA levels of MCP-1 (r = 0.56, P<0.05), osteopontin (r = 0.50, P<0.05) and TGF-ß1 (r = 0.53, P<0.05) in the renal cortex of OLETF rats.
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Discussion |
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In previous studies, macrophage infiltration was promoted by the increased expression of intercellular adhesion molecules (ICAM)-1 [14], E- and P-selectin [2], and MCP-1 [6,7] in diabetic patients and in animal models. Osteopontin upregulation is also important for renal damage, especially tubulointerstitial injuries, by promoting interstitial macrophage accumulation in diabetic kidneys [8]. We showed that MCP-1 and osteopontin expression in the kidneys of OLETF rats was significantly increased compared with that in LETO rats. Although the mechanisms of upregulation for these molecules are not clear, they are thought to be induced by high ambient glucose levels, high shear stress and an activated RAS. Treatment with an ACEI or ARB has been shown to suppress renal MCP-1 and osteopontin expression and to attenuate renal injury without altering glycaemic control in diabetic rats [7,8]. These drugs may suppress AGE formation and accumulation, resulting in the progression of diabetic renal injury in the kidneys [15]. Therefore, the use of these agents at the early stage of human diabetic nephropathy is recommended. However, once overt nephropathy has begun, improvement in proteinuria or protection against progressive renal injury is difficult to achieve using only glycaemic control and RAS blockade in many diabetic patients. For these patients, superior therapeutic agents are required.
It was reprorted recently that MMF, an anti-inflammatory agent, provides treatment against streptozotocin-induced diabetic nephropathy [9]. For example, a study showed that MMF suppressed glomerular macrophage accumulation and glomerulosclerosis in uninephrectomized diabetic kidneys without affecting control of hyperglycaemia or hypertension. Mizoribine acts through similar mechanisms to MMF to cause immunosuppression [10]. Therefore, mizoribine has seen frequent use in patients with lupus nephritis and primary nephrotic syndrome in Japan. The adverse effects of mizoribine such as myelosuppression, hepatotoxicity and nephrotoxicity are rare compared with other immunosuppressive agents [11]. Indeed, administration of 10 mg/kg of mizoribine did not exert any adverse effects in OLETF rats. Such findings indicate that mizoribine may be of great clinical advantage. Mizoribine also inhibited tubular osteopontin expression, interstitial macrophage infiltration and interstitial fibrosis in rats with unilateral ureteral obstruction, a non-immunological renal damaged model [16]. We showed that mizoribine inhibited production of MCP-1, osteopontin and TGF-ß1, that it suppressed renal macrophage infiltration and that it improved glomerulosclerosis, interstitial injury and urinary albumin excretion in OLETF rats. The precise mechanisms by which mizoribine prevented the progression of diabetic nephropathy are not known. However, it could inhibit the upregulation of chemokines such as MCP-1 and osteopontin, as well as suppress the proliferation of macrophages and lymphocytes. Because macrophage accumulation in the kidney was suppressed, the production of TGF-ß1 may also have been inhibited, resulting in prevention of diabetic nephropathy progression. The positive correlations between the number of interstitial ED1-positive cells and mRNA levels of these molecules in the kidneys may support this hypothesis.
In the present study, the renoprotective effect of mizoribine against diabetic nephropathy appeared to be modest compared with that of MMF. This finding may be explained by specific characteristics of our present experiments. First, we used a type 2 diabetic model that was complicated by a variety of metabolic disorders other than hyperglycaemia and hypertension. Secondly, mizoribine was administered to rats with overt diabetic nephropathy for a short duration, whereas MMF was administered to rats not having nephropathy over a long time period [9]. We believe that these drugs should not be used to protect against nephropathy in diabetic subjects because this patient group, especially those having poor glycaemic control, may be in immunosuppressed states. We therefore used OLETF rats at 35 weeks of age, which already had increased urinary albumin excretion and showed pathological changes equal to stages 23A in human diabetic nephropathy. Male OLETF rats usually show hyperglycaemia from 25 weeks of age and have increased urinary protein from 30 weeks of age. The OLETF rats that we examined in this study showed a similar evolution of diabetes. Importantly, we found that an anti-inflammatory drug was effective even in type 2 diabetic kidneys complicated by hyperlipidaemia, hypertension and obesity. Renoprotective effects of ACEIs have been reported in OLETF rats [8,17,18]. However, ACEIs were administered to rats during early stages of nephropathy (2022 weeks of age) for a longer time period (28 weeks to 9 months). In these studies, the effect of enalapril [17] and imidapril [18] on albuminuria in OLETF rats was significant but inadequate. Although the effect of these drugs on rats with overt diabetic nephropathy is unknown, the finding that short-term treatment with mizoribine inhibited progression of moderate stage nephropathy in non-insulin-dependent diabetic rats is of potential clinical importance.
It is clear that intensive glycaemic control is very important in protecting against nephropathy in type 2 diabetic patients, and this has been supported by the United Kingdom Prospective Diabetes Study (UKPDS) [19] and the Kumamoto Study [20]. However, once overt proteinuria appears, glycaemic control alone cannot inhibit the progression of nephropathy. As previously mentioned, RAS blockade was shown to slow, both experimentally and clinically, the progression of nephropathy and to improve renal survival in diabetic patients. However, the use of a single drug in the current study did not completely suppress diabetic renal injuries. Whether anti-inflammatory drugs combined with RAS blockade have additional renoprotective effects against diabetic nephropathy requires further examination.
In conclusion, mizoribine inhibited glomerular and interstitial macrophage accumulation and improved glomerulosclerosis and interstitial fibrosis in non-insulin-dependent diabetic kidneys. Combination therapy with anti-inflammatory agents such as mizoribine and MMF coupled with glycaemic control and RAS blockade may be useful in the management of non-insulin-dependent diabetic nephropathy with massive proteinuria.
Conflict of interest statement. None declared.
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References |
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