The incidence of end-stage renal disease (ESRD) is a growing problem.1,2 In most countries, diabetes is the primary cause of ESRD in patients who receive ESRD therapy. Diabetic nephropathy occurs in 20-40% of patients with diabetes and is the single leading cause of ESRD.3 Because most renal disease ultimately progresses to ESRD, the emphasis of treatment focuses on the prevention of disease progression.
Systemic hypertension accelerates the progression of diabetic nephropathy, whereas lowering blood pressure reduces renal damage. The current strategy for treating patients with diabetes recommends the use of well-tolerated antihypertensive agents, particularly angiotensin converting enzyme inhibitors and angiotensin type 1 (AT1) receptor blockers (ARBs). Recent clinical trials have shown that treatment with ARBs leads to an improvement in renal outcomes in patients with type 2 diabetes.4-7
The renal protective effects of ARBs have been demonstrated in animal models of diabetes, including type 1 and type 2 diabetic rats.8,9 We also have reported that olmesartan medoxomil (OLM),10 an ARB, administered from the early phase of diabetic nephropathy with albuminuria, suppressed the progression of diabetic nephropathy in Zucker Diabetic Fatty (ZDF) rats, a model of type 2 diabetes.11 In most of the animal studies, however, the drug was administered from the early phase of proteinuria in order to examine the effects on progression of diabetic nephropathy. The effect of ARBs, administered from the advanced phase of diabetic nephropathy, has not been investigated. In the present study, OLM was administered after the onset of overt proteinuria in ZDF rats and the effects on diabetic nephropathy and mortality were evaluated. To explore the mechanism underlying the renoprotective effect of OLM, we examined monocyte/macrophage infiltration and monocyte chemoattractant protein-1 (MCP-1) staining in the kidneys because MCP-1 has been known to recruit and activate monocytes to induce inflammation. We also conducted in vitro experiments with cultured cells to interpret the results of in vivo experiments.
MATERIALS AND METHODS
All experiments were carried out in accordance with the Animal Experimentation Guidelines of Sankyo Co., Ltd. Male ZDF rats (ZDF/Gmi-fa/fa) and male nondiabetic lean rats (ZDF/Gmi-+/+ or -+/fa, mixed genotype) were purchased from Charles River Japan, Inc. The rats were kept in a room at 55% relative humidity (permissible range: 30-70%) and 23°C (permissible range: 20-26°C) under a 12-hour light/dark cycle. They were allowed free access to diet (FR-2, Funabashi Farm Co., Ltd.) and water.
At 36 weeks of age, the ZDF rats were allocated by body weight, plasma glucose, urinary protein excretion, and systolic blood pressure to the following two groups: the untreated ZDF group and the OLM-treated ZDF group. In the latter group, OLM was mixed in the diet at a concentration of 0.01%. Based on food consumption, the average dose of OLM was estimated to be about 6.4-8.0 mg/kg/day during drug administration. Drug administration was continued until 63 weeks of age, when the survival rate for the untreated ZDF became less than 50%. The lean rats and the untreated ZDF rats were fed a normal drug-free diet.
Biochemical and Blood Pressure Measurement
Urine and blood samples were collected every 2 or 4 weeks over 14-62 weeks of age. Systolic blood pressure was usually measured the week after the collection of the urine samples.
To collect the urine samples, rats were placed in individual metabolic cages for 24 hours. After measurement of the volume, the urine samples were centrifuged at 4°C and 3,000 rpm for 10 min. A supernatant was used for assays of the urinary protein concentration (Protein Assay Rapid Kit, Wako Pure Chemical Industries, Ltd.).
The blood samples were withdrawn from the tail vein into heparinized micro test tubes. Plasma was obtained by centrifuging the blood samples at 4°C and 3,000 rpm for 20 min. The plasma samples were used for measurements of creatinine concentration (Creatinine Test-WAKO, Wako Pure Chemical Industries, Ltd.), glucose (Glucoroder-F, A&T Corporation), blood urea nitrogen (BUN) (Model 7250, Hitachi, Ltd.), lipid concentration (Model 7250, Hitachi, Ltd.), and albumin (Model 7250, Hitachi, Ltd.).
Systolic blood pressure was measured noninvasively with an autosphygmomanometer (BP-98A, Softron Corp.).
Histology and Immunohistochemistry
Following the final measurements of the biochemical parameters and blood pressure, the surviving animal was sacrificed and the kidney was excised, fixed in 10% formalin, and embedded in paraffin. Microscope sections were prepared and stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and periodic acid methenamine silver (PAM) for the histological examination. Most of the kidney preparations from the animals that died during the experimental period showed low intensity of staining, probably because of spoilage of the tissue. For this reason, only the data from the surviving animals were used for histological analysis. Glomerular sclerosis, tubular damage, and arterial injury were semiquantitatively evaluated under microscopic observation according to the criteria developed by Uehara et al.12 For the immunohistochemical examination, a paraffin-embedded section was incubated with a primary anti-MCP-1 antibody (AbCam, Ltd.) or anti-ED1 antibody (Serotec, Ltd.). To detect the primary antibody, the section was incubated with biotin-conjugated secondary antibody (DakoCytomation A/S), then incubated with streptavidin-peroxidase solution using an ABC Kit (Vector Laboratories, Inc.) and, lastly, stained with diaminobenzidine (DakoCytomation A/S). Positively stained glomeruli were counted under the microscope.
Detection of MCP-1 Release from Cultured Tubular Epithelial Cells and Mesangial Cells
Cultured human tubular epithelial cells were incubated with human serum albumin at a concentration of 5 mg/mL, with or without angiotensin II (AII) at a concentration of 100 nM in the presence or absence of olmesartan, the active form of OLM, at a concentration of 1 μM for 24 hours. After incubation, culture medium of tubular cells was collected for measurement of the MCP-1 concentration by enzyme-linked immunosorbent assay (R&D System, Inc.). In other experiments, cultured human mesangial cells were incubated with AII at a concentration of 100 nM, with or without olmesartan at a concentration of 1 μM, for 24 hours. The MCP-1 concentration in the culture medium of mesangial cells was measured by enzyme-linked immunosorbent assay as described above.
All data were expressed as the means ± SEM. The statistical difference between the lean group and the untreated ZDF group was determined by the Student t-test. The statistical difference between the untreated and OLM-treated ZDF groups was also determined by the t-test. The survival rate was analyzed using the Kaplan-Meier method, following the log-rank analysis and the hazard ratio calculation. The Wilcoxon rank sum test was used to compare the semiquantitatively measured histological data. All calculations were done using SAS System Release 8.2 (SAS Institute Inc.). The value of P < 0.05 was considered statistically significant.
The ZDF rats were heavier than the lean rats at 14 weeks of age and remained heavier up to 22 weeks of age, but were not significantly different in body weight from the lean rats at 26 to 34 weeks of age. At 38 weeks of age and thereafter, the ZDF rats were slightly lighter than the lean rats. Treatment with OLM did not affect the body weight in the ZDF rats at any measurement week (data not shown).
Plasma Glucose Concentration
The plasma glucose concentration of the ZDF rats at the age of 34 weeks was higher than that of the lean rats (155 ± 3 mg/dL and 519 ± 13 mg/dL in the lean rats and ZDF rats, respectively; P < 0.001). The plasma glucose concentration of the untreated ZDF rats increased with age until 54 weeks and declined thereafter. It was significantly higher than that of the lean rats throughout the experimental period. OLM did not decrease the plasma glucose concentration in the ZDF rats at any measurement week (515 ± 25 mg/dL and 448 ± 54 mg/dL in OLM-treated ZDF rats and untreated ZDF rats at 62 weeks of age, respectively; P > 0.05).
Systolic Blood Pressure
Systolic blood pressure of the ZDF rats was not different from that of the lean rats up to 39 weeks of age (136 ± 2 mmHg and 140 ± 2 mmHg in the lean rats and ZDF rats at 39 weeks of age, respectively; P > 0.05), but it was slightly higher than that of the lean rats at 43 weeks of age and thereafter. OLM significantly lowered blood pressure throughout the treatment period in the ZDF rats (124 ± 3 mmHg and 158 ± 7 mmHg in OLM-treated and untreated ZDF rats at 63 weeks of age, respectively; P < 0.001).
Urinary Protein Excretion
Figure 1A shows the effect of OLM on urinary protein excretion in ZDF rats. Urinary protein excretion in the ZDF rats increased with age and was significantly greater compared with that of the lean rats. Dosing of OLM was started at the age of 36 weeks when the ZDF rats exhibited massive proteinuria (698 ± 31 mg/kg/day and 29 ± 2 in the ZDF rats and the lean rats at the age of 34 weeks, respectively; P < 0.001; Fig. 1A). OLM dramatically retarded or stopped the progression of proteinuria in the ZDF rats throughout the entire treatment period. At the end of the dosing period, urinary protein excretion in the surviving untreated ZDF rats (N = 7) was 2297 ± 207 mg/kg/day versus 854 ± 137 mg/kg/day in surviving OLM-treated ZDF (N = 21) (P < 0.001).
Plasma Albumin Concentration
Figure 1B shows the effect of OLM on plasma albumin concentration in ZDF rats. Plasma albumin concentration in the ZDF rats was significantly lower than that in the lean rats at 16 weeks of age and remained lower throughout the experimental period. OLM significantly inhibited the decrease in plasma albumin concentration in the ZDF rats at 38 weeks of age and thereafter.
Plasma Lipid Concentrations
Plasma total cholesterol concentration in the ZDF rats was significantly higher than that in the lean rats (data not shown). Plasma triglyceride, high-density lipoprotein cholesterol, free cholesterol, and phospholipid concentrations of the ZDF rats were also significantly higher than those of the lean rats (data not shown). OLM significantly lowered these plasma lipid concentrations at 38 weeks of age and thereafter. Plasma nonesterified fatty acid concentration was not different among the groups (data not shown).
BUN and Plasma Creatinine Concentration
An abrupt increase in BUN was observed in the untreated ZDF group at 50 weeks of age and thereafter. OLM suppressed the increase in BUN in the ZDF rats (Fig. 2A). An abrupt increase in plasma creatinine concentration was also observed in the untreated ZDF rats. OLM significantly inhibited the increase in plasma creatinine concentration as well (data not shown). The slope of 1/creatinine over time was calculated as an index of renal function decline (Fig. 3). The slope of 1/creatinine over time in untreated ZDF rats was -0.0566 dL/mg/week, which was almost identical to that in OLM-treated ZDF rats before the start of dosing. Of note is that the slope turned to a positive value (slope = 0.0219 dL/mg/week) after treatment with OLM (Fig. 3B).
Figure 2B shows the effect of OLM on the survival rate of ZDF rats. The ZDF rats started to die at 50 weeks of age in the untreated group, and the survival rate decreased sharply from 54 weeks of age. Death was generally preceded by an increase of plasma creatinine concentration and BUN, suggesting that the cause of death was severe renal dysfunction. Although dead rats were also observed from 53 weeks of age in the OLM-treated ZDF group, the survival rate at 62 weeks of age was much higher in the OLM-treated ZDF group (80%) than in the untreated ZDF group (32%) (log-rank test, P = 0.0004; hazard ratio for mortality, 0.191; 95% confidence interval [CI]: 0.069-0.531).
Histology and Immunohistochemistry
Figure 4 shows photomicrographs which exhibit examples of tubular, glomerular, and arterial injuries. The left side photos (Fig. 4A, D, and G) are from lean rats, the middle ones (Fig. 4B, E, and H) are from untreated ZDF rats, and the right ones (Fig. 4C, F, and I) are from OLM-treated ZDF rats. Extensive glomerular sclerosis, tubular injury, and arterial injury were observed in the untreated ZDF rats, but not in the lean control rats. These pathological changes were ameliorated by the treatment with OLM (Table 1, Fig. 4).
Figure 5 shows MCP-1 protein expression (Fig. 5A-C) and macrophage infiltration (Fig. 5D-F) in the kidney. The MCP-1 protein was detected in the tubular cells of the untreated ZDF rats. This expression was more clearly visible in undilated tubular cells than in dilated tubular cells, suggesting that seriously injured tubular cells lose the capacity to produce MCP-1. Some of the glomeruli of the untreated ZDF rats had a positive expression of MCP-1 protein. Macrophage infiltration, which was detected by ED1-positive staining, was observed in the tubulointerstitium and glomeruli in the untreated ZDF rats, but was decreased by the treatment with OLM. Table 1 shows the percentages of glomeruli positively stained for MCP-1 and ED1. The percentages of MCP-1 and ED1-positive glomeruli were greater in the untreated ZDF rats compared with the lean rats and were decreased by treatment with OLM. Figure 6 shows the correlations between urinary protein excretion and glomerular macrophage infiltration or glomerular MCP-1 expression. Urinary protein excretion was significantly correlated with glomerular MCP-1 expression (Fig. 6A; r = 0.663, P < 0.001) and also with glomerular macrophage infiltration (Fig. 6B; r = 0.630, P < 0.001).
MCP-1 Release from Cultured Tubular Epithelial Cells and Mesangial Cells
In a separate series of in vitro experiments, human serum albumin markedly increased MCP-1 release from cultured human tubular epithelial cells (Fig. 7A). AII did not affect basal MCP-1 release nor the MCP-1 release induced by human serum albumin. Olmesartan, the active form of OLM, also did not influence the effect of human serum albumin on MCP-1 release in human tubular epithelial cells (data not shown). In contrast, AII slightly but significantly increased MCP-1 release from isolated cultured human mesangial cells (Fig. 7B). Olmesartan inhibited the basal MCP-1 release and also MCP-1 release induced by AII in cultured human mesangial cells.
In the present study, OLM, when administered from the advanced phase of diabetic nephropathy, retarded progression of the disease, corrected abnormal lipid metabolism, and increased the survival rate in ZDF rats. OLM also changed the slope of 1/creatinine over time, an index of renal function decline. A reversal of the slope from a negative value to a positive value after the start of OLM treatment indicates that the drug not only stopped the worsening of renal function but improved it. This fact suggests that ARB, when used in appropriate doses, has a capacity to restore once deteriorated kidney function. To our knowledge, there has been no demonstration in either human studies or animal studies that an RAS blocker, when administered from the advanced stage of diabetic nephropathy, can restore the renal function.
Recently, the effect of irbesartan on the survival of obese Zucker rats, which is different from ZDF rats used in the present study, was reported.13 In this report, treatment with irbesartan normalized hyperglycemia and delayed the onset of mortality associated with diabetic nephropathy in obese Zucker rats. Because antidiabetic agents such as rosiglitazone are reported to lower the plasma glucose level and inhibit proteinuria,14 it is not clear whether the renoprotective effect of irbesartan on diabetic nephropathy is due to a hypoglycemic effect or an AII blocking effect. In our study, the amelioration of renal disease was not accompanied by the lowering of either plasma glucose or insulin concentrations, suggesting that the renoprotective effect of OLM on diabetic nephropathy is not dependent on glucose metabolism. This finding is in accord with the results of the RENAAL (Reduction of Endpoints in NIDDM with an Angiotensin II Antagonist Losartan) substudy in which losartan reduced the incidence of ESRD, but did not affect the HbA1c level.15
MCP-1 has been known as a chemokine that stimulates monocyte/macrophage infiltration and it has been implicated in interstitial inflammation. In order to explore the mechanism underlying the beneficial effect of OLM on diabetic nephropathy, we examined monocyte/macrophage infiltration and MCP-1 expression in the kidney. In the present study with ZDF rats, we found that OLM decreased MCP-1 in the tubular cells and reduced macrophage infiltration in the interstitial space (Fig. 5). In the in vitro experiments with human tubular epithelial cells, we demonstrated that albumin increased MCP-1 production in the tubular cells and that this was not affected by treatment with AII or the active form of OLM (Fig. 7). MCP-1 produced by the tubular cells would be distributed into the interstitial space to induce interstitial inflammation. Accordingly, plasma albumin leaked from the glomeruli would induce inflammatory changes via enhanced expression of MCP-1 in the tubular interstitium. This notion is supported by other investigators as well.16,17 Our observations in in vivo and in vitro studies suggest that OLM reduced MCP-1 production and macrophage infiltration in the tubular interstitium by suppressing albumin leakage from glomeruli. In other words, the suppression of proteinuria would play a pivotal role in renal protection by an AT1 receptor blockade.
We also focused on the macrophage infiltration in the glomerulus. AII increased the release of MCP-1 from cultured mesangial cells in the present study. Although plasma renin activity is decreased in diabetes mellitus,18 the renin-angiotensin system is activated in the renal tissue of diabetic rats.19,20 Consistent with this fact, high glucose has been reported to increase AII generation in cultured mesangial cells.21 Taken together, these facts suggest that increased AII generation causes macrophage infiltration in the glomerulus via enhanced MCP-1 expression. OLM might decrease macrophage infiltration in the glomerulus by interrupting this sequence. In fact, we demonstrated that both MCP-1 expression and macrophage infiltration in the glomerulus correlated with urinary protein excretion in the ZDF rats (Fig. 6) and that these histological changes were corrected by the treatment with OLM (Fig. 4 and Table 1).
OLM decreased MCP-1 expression in both the glomeruli and tubules and this might be important for the antidiabetic nephropathy effects of OLM. However, the mechanisms for the decrease in MCP-1 expression are different between the glomeruli and tubules: a direct AT1 receptor blockade would be the primary cause in glomerular cells and an indirect effect via reduction of proteinuria might be a major path in tubular cells.
In summary, OLM, when administered from the advanced stage of diabetic nephropathy with massive proteinuria, delayed the progression of nephropathy and increased the survival rate in ZDF rats, a model of type 2 diabetes. The results of in vivo and in vitro experiments suggest that decrease of protein excretion plays a pivotal role in the renal protective action of AT1 receptor blockade.
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