Long-Term Treatment of Glucagon-Like Peptide-1 Analog Exendin-4 Ameliorates Diabetic Nephropathy through Improving Metabolic Anomalies in db/db Mice

Abstract

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-β1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator–activated receptor α and GLP-1 receptor–positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.

In addition to the prominent role played by hyperglycemia, hypertension, growth factors (including angiotensin II, TGF-β, connective tissue growth factor, and vascular endothelial growth factor), inflammatory cytokines, oxidative stress, and advanced glycation end products, abnormal lipid metabolism as well as renal accumulation of lipids have been proposed to play a role in the pathogenesis of diabetic nephropathy (^1,2). A recent study demonstrated that in diabetes, hyperglycemia per se directly upregulates renal expression of the transcriptional factor sterol regulatory element-binding protein-1, which caused increased fatty acid synthesis and accumulation of triglycerides. This was associated with upregulation of TGF-β and vascular endothelial growth factor expression and accumulation of collagen and fibronectin, which result in mesangial expansion, glomerulosclerosis, and proteinuria (³).

Accumulation of excess lipids in nonadipose tissues leads to cell dysfunction or cell death (^4,5). This phenomenon, known as lipotoxicity, may play an important role in the pathogenesis of diabetes and heart failure in humans (⁴). Several mechanisms have been demonstrated to underlie lipotoxicity, including a direct toxic effect of fatty acids or products of their metabolism, increased production of reactive oxygen species, ATP deficiency, and fatty acid–induced apoptosis (^4–7). Our previous studies demonstrated that the activation of peroxisome proliferator–activated receptor-α (PPAR-α) by fenofibrate improves diabetic nephropathy in db/db mice, and the lack of the PPAR-α accelerates diabetic nephropathy in streptozotocin-induced type 1 diabetes (^6,7).

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and also considered a potential therapeutic agent for type 2 diabetes because it stimulates β cell proliferation and insulin secretion in a glucose-dependent manner, inhibits glucagon secretion, induces satiety, and delays gastric emptying, which together result in reduced circulating glucose (^8,9). However, this peptide is almost immediately degraded by dipeptidyl peptidase-IV. In a clinical study, a group of patients who had type 2 diabetes and received GLP-1 as a continuous subcutaneous infusion for 6 wk showed that fasting and average plasma glucose concentrations were lowered by approximately 5 mmol/L and free fatty acids (FFA) were significantly lowered, and the patients lost 2 kg in weight (¹⁰). Furthermore, insulin sensitivity and insulin secretion capacity greatly improved. Despite the marked metabolic improvement, plasma glucose levels were not completely normalized.

Exendin-4 is a 39–amino acid peptide that originally was isolated from the salivary secretions of the Gila monster lizard (¹¹). It shares approximately 53% homology with the mammalian incretin GLP-1 and binds to and activates the mammalian receptor for GLP-1 cloned from pancreatic β cells (^12,13). These studies examined the effect of GLP-1, using the long-acting GLP-1 analog exendin-4, on the development and progression of diabetic nephropathy in db/db mice.

Materials and Methods

Experimental Methods

All experiments were performed according to institutional animal care guidelines. Six-week-old male C57BLKS/J db/db and db/m mice were purchased from Jackson Laboratories (Bar Harbor, ME); db/m mice were used as controls in all experiments. Mice were maintained on a 12-h light/dark cycle and were fed a standard laboratory diet and water ad libitum in a room that was controlled for temperature (23 ± 3°C) and humidity (55 ± 15%). Exendin-4 (Sigma, St. Louis, MO) in 0.5 or 1.0 nmol/kg per d (0.5 or 1.0 exendin-4, respectively) was administrated intraperitoneally to db/db mice (n = 8, respectively) and age- and gender-matched db/m mice (n = 7) for 8 wk starting at age of 8 wk. Control db/db mice (n = 8) and control db/m mice (n = 6) received saline for 8 wk.

For measurement of 24-h albumin excretion and creatinine clearance, mice were placed in individual mouse metabolic cages (Nalgene, Rochester, NY) with access to water and food for 24 h. Mouse body weight was measured weekly. Blood glucose was measured every 2 wk, and glycated hemoglobin (HbA_1c) and 24-h urinary albumin and creatinine were measured every 4 wk. At 8 wk, intraperitoneal glucose tolerance and systolic BP were assessed by a noninvasive tail-cuff system in conscious mice at the end of the study (IITC Life Science, Woodland Hills, CA). Mice were habituated to the tail-cuff device before measurement of the BP for 5 d over 15 min to reduce variability of the measurements. After 8 wk, mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (55 mg/kg body wt; Nembutal; Boehringer Ingelheim, Artarmon, NSW, Australia). The kidneys were rapidly dissected and stored in buffered formalin (10%) for subsequent immunohistochemical analyses.

Measurement of Serum Parameters

Blood was collected from the left ventricle and centrifuged, and plasma was stored at −70°C for subsequent analyses. HbA_1c was determined on red cell lysates by HPLC (BioRad, Richmond, CA). Total cholesterol, triglyceride, FFA, and insulin concentrations were measured by autoanalyzer (Wako, Osaka, Japan). Homeostasis model assessment for insulin resistance (HOMA_IR) index was calculated as follows: Fasting glucose (mmol/L) × fasting insulin (mU/L)/22.5.

Assessment of Renal Function

At weeks 0, 4, and 8, the mice were housed in metabolic cages for 24 h to collect urine for subsequent measurements of albumin concentration by an immunoassay (Bayer, Elkhart, IN). At week 8, plasma and urinary creatinine and urea concentrations were measured using HPLC and autoanalyzer (Beckman Instruments, Fullerton, CA), respectively. Creatinine clearance was calculated by (urine [Cr] × urine volume)/(plasma [Cr] × time).

Light Microscopic Study

Kidney and liver samples were collected after systemic perfusion with PBS and then fixed in 4% paraformaldehyde. Histology was assessed after hematoxylin and eosin and periodic acid-Schiff staining. To examine the effect of exendin-4 on glomerular area and mesangial matrix area, we performed glomerular analysis on periodic acid-Schiff–stained kidney sections. Mesangial matrix area and glomerular tuft area were quantified for each glomerular cross-section as previously reported (^6,7). More than 30 glomeruli that were cut through the vascular pole were counted per kidney, and the average was used for analysis. Furthermore, to evaluate the effect of exendin-4 on lipid accumulation in the glomerulus, we performed oil red O on staining frozen renal tissue.

Immunohistochemistry for TGF-β1, Type IV Collagen, F4/80, Caspase-3, PPAR-α, GLP-1R, and 8-Hydroxy-Deoxyguanosine

We performed immunohistochemistry for type IV collagen, TGF-β1, F4/80, caspase-3, PPAR-α, and 8-hydroxy-deoxyguanosine (8-OH-dG). Briefly, small blocks of kidney were immediately fixed in 10% buffered formalin for 24 h before being embedded in paraffin. Five-micrometer-thick sections were deparaffinized, washed with PBS, and incubated with 1.5% H₂O₂ in methanol to block endogenous peroxidase activity. Nonspecific binding was blocked with 10% normal goat serum in PBS. Sections were incubated overnight with the anti–type IV collagen (1:150 in blocking solution; Biodesign Int., Saco, ME), anti–TGF-β1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-F4/80 (1:100; Serotek, Oxford, UK), anti–caspase-3 (1:50; Santa Cruz Biotechnology), anti–PPAR-α (1:1500; provided by Dr. A. Sugihara, Tokohu University Graduate School of Medicine, Sendai, Japan), GLP-1R (1:200; Abcam, Kendall Square, MA), and 8-OH-dG (1:100; JalCA, Shizuoka, Japan) in a humidified chamber at 4°C. Tissue sections were treated with an antigen-unmasking solution that consisted of 10 mM Na citrate (pH 6.0) and 0.05% Tween 20. Antibodies were localized with the ABC technique (Vector Laboratories, Burlingame, CA) and 3,3-diaminobenzidine substrate solution with nickel chloride enhancement. Sections were then dehydrated in ethanol, cleared in xylene, and mounted without counterstaining.

All of these sections were examined in a masked manner using light microscopy (Olympus BX-50; Olympus Optical, Tokyo, Japan). For the quantification of proportional area of staining, approximately 20 views (×400 magnification) were randomly located in the renal cortex and corticomedullary junction of each slide (Scion Image Beta 4.0.2, Frederick, MD).

24-Hour Urinary 8-OH-dG

To determine the oxidative DNA damage in the kidney, we determined 24-h urinary 8-OH-dG concentrations using competitive ELISA (8-OH-dG Check; Institute for the Control of Aging, Shizuoka, Japan).

Measurement of Liver and Kidney Lipids

Liver and kidney lipid contents were measured using assay kits from Waco Co., (Osaka, Japan). Liver and kidney lipids were extracted by the method of Bligh and Dyer with slight modifications (¹⁴). A portion (50 mg) of the liver or kidney was homogenized and extracted by methanol-chloroform aliquots (2:1) in a 37°C water bath under N₂. The lower chloroform phase was withdrawn, and lipids were measured using the assay kits.

Statistical Analyses

Data are expressed as means ± SD. Differences between the groups were examined for statistical significance using ANOVA with Bonferroni correction (SPSS 11.5, Chicago, IL). P < 0.05 was considered a statistically significant difference.

Results

Body Weight, Liver Weight, Peri-Epididymal Fatty Tissue Weight, and the Levels of Glucose and HbA_1c

Body weight of db/db mice that were treated with 0.5 or 1.0 exendin-4 was less than that of control db/db mice at the end of experimentation (P < 0.05; Figure 1A). There was no such difference between db/m mice that were treated with or without exendin-4. There was significant decrease in body weights in db/db mice that were treated with 0.5 or 1.0 exendin-4 (P < 0.05, respectively; Figure 1B) compared with those of control db/db mice. The weight of peri-epididymal fat of db/db mice that were treated with 0.5 or 1.0 exendin-4 was significantly decreased compared with control db/db mice (P < 0.01 and P < 0.001, respectively; Table 1). There was no difference in liver weight and food intake among db/db mice that were treated with or without exendin-4 (Table 1). Diabetic db/db mice exhibited dramatically increased blood glucose and HbA_1c levels throughout the entire period of the experiment (P < 0.001; Figure 2) compared with those of nondiabetic db/m mice. However, there were no significant differences in blood glucose and HbA_1c among db/db mice.

Blood Chemistry, Serum Lipid and Insulin Levels, and HOMA_IR Index

There was no difference in hematocrit among diabetic db/db mice with or without exendin-4 treatment (Table 1). In contrast, serum FFA and triglyceride levels were significantly decreased in db/db mice that were treated with 1.0 exendin-4 compared with those of control db/db mice (P < 0.05; Table 2). Furthermore, serum insulin concentrations (P = 0.05 and P = 0.001, respectively) and HOMA_IR indices (P = 0.011 and P < 0.001, respectively) were markedly decreased in db/db mice that were treated with exendin-4 in a dosage-dependent manner (Table 2). There was no such difference between db/m mice that were treated with or without exendin-4. It is interesting that there were no differences in total cholesterol concentrations among the groups (Table 2).

Kidney Weight, Albuminuria, Serum Creatinine, Creatinine Clearance, and Systolic BP

The kidney weight, blood urea nitrogen, and creatinine in blood did not differ significantly between experimental groups (Table 3). As expected, db/db mice exhibited an increase in 24-h urine volumes; 1.0 exendin-4 treatment in db/db mice profoundly decreased urine volume (P < 0.01; Table 3), resulting from decreased water intake (P = 0.03; Table 1). Diabetic db/db mice also exhibited a persistent increase in creatinine clearance and urine albumin excretion, whereas creatinine clearance by HPLC and albuminuria in exendin-4–treated db/db mice decreased to the levels of db/m mice in a dosage-dependent manner (P < 0.05 and P = 0.005, respectively; Table 3). No significant differences in systolic BP (Table 1) were observed among the groups.

Liver and Renal Histologic Examination

In db/db mice, severe macrovesicular fatty liver was prominent in the whole liver; however, exendin-4 treatment markedly improved hepatic steatosis in a dosage-dependent manner (Figure 3). Glomerular injury in diabetic db/db mice was characterized by mesangial extracellular matrix expansion and glomerulosclerosis (Figure 4, A through E). Increased glomerular fractional mesangial areas were decreased in db/db mice that were treated with 1.0 exendin-4 (P < 0.01; Figure 4F), but there was no difference between glomerular fractional mesangial ares in db/db mice and those of 0.5 exendin-4–treated db/db mice.

Expression of TGF-β1, Type IV Collagen, and Macrophage Infiltration

Diabetes was associated with an increase in TGF-β1 expression in the glomerulus (Figure 4G). In the kidneys of exendin-4–treated db/db mice, positive staining for TGF-β1 was significantly decreased and localized to the glomerulus in a dosage-dependent manner (P < 0.05 and P < 0.01, respectively; Figure 4, H and I). In contrast, there was no such change in db/m mice (Figure 4, J, K, and L). Diabetes was also associated with an increase in type IV collagen protein expression in the glomerulus (Figure 4, M through Q). The prominent increase in type IV collagen in the glomerulus in control db/db mice was markedly decreased in exendin-4–treated db/db mice in a dosage-dependent manner (P < 0.05 and P < 0.01; Figure 4R).

Only modest macrophage infiltration, as assessed by F4/80-positive staining, was observed in the glomerulus of db/m mice with or without exendin-4 treatment (Figure 4, S through W). In contrast, F4/80 immunostaining was markedly increased in the glomerulus of db/db mice compared with that of db/m mice (Figure 4S). Exendin-4 treatment significantly decreased the expression of F4/80 immunostaining in db/m mice in a dosage-dependent manner as well (Figure 4, V and X).

Expression of Caspase-3

In the kidneys of nondiabetic db/m mice, a few caspase-3–positive cells were observed within the glomeruli (Figure 5, A through E). In db/db mice, diabetes was associated with a markedly increased number of glomerular caspase-3–positive cells compared with those of other db/db mice groups (P < 0.001; Figure 5A). In contrast, when db/db mice were treated with exendin-4, especially 1.0 exendin-4, caspase-3 immunostaining was markedly decreased (P < 0.001; Figure 5, E and F).

Expression of PPAR-α and Lipid Staining in the Glomerulus

Immunohistochemistry revealed significantly increased PPAR-α expression in exendin-4–treated db/db kidney compared with that in control db/db kidneys (Figure 6). The increases in renal PPAR-α immunostaining in the db/db mice that were treated with 0.5 or 1.0 exendin-4 by 1.5- or 2.5-fold, respectively, compared with control db/db mice occurred primarily in the proximal tubules and thick ascending tubules (Figure 6, B and C). Oil red O staining of kidney sections showed no apparent lipid deposition in the kidneys of control db/m mice regardless of the treatment of exendin-4 (Figure 7, D and E). Conversely, in db/db mice, marked accumulation of oil red O–stainable lipid was observed in the glomerular and tubular epithelial cells in kidney section (Figure 7A). Only a small fraction of lipid droplets were found in the glomerulus in db/db mice that were treated with both 0.5 and 1.0 exendin-4, respectively (Figure 7, B and C).

Effect of Exendin-4 on 24-H Urinary Concentrations, Immunostaining of 8-OH-dG, and GLP-1R

Treatment with 1.0 exendin-4 decreased markedly 24-h urinary 8-OH-dG in db/db and db/m mice as determined by ELISA (Figure 8A). To evaluate further the effect of exendin-4 on the kidney, we performed immunostaining of 8-OH-dG expression in the kidney. In the glomerulus, strongly increased immunostaining expression of 8-OH-dG (dark brown nucleus) in db/db mice markedly attenuated by treatment with 1.0 exendin-4 (Figure 8C) compared with control db/db mice (Figure 8B). However, there was no difference in 8-OH-dG immunostaining in db/m mice with or without exendin-4 treatment (Figure 8, D and E). To determine the effect of exendin-4 on GLP-1R expression in the glomerulus, we also performed immunostaining for GLP-1R in the kidney. Noticeably increased GLP-1R–positive cell expression was seen in the glomeruli of db/db mice that were treated with 0.5 (P < 0.001 compared with db/db control; Figure 9, A and B) or 1.0 exendin-4 (P < 0.001 compared with 0.5 exendin-4–treated db/db mice; Figure 9C). In db/m mice, the expressions of GLP-1R–positive cells was evidently increased in db/m compared with exendin-4–treated db/db mice (P < 0004 compared with 1.0 exendin-4 db/db [Figure 9D]; P < 0.001 compared with db/m control [Figure 9E]).

Effect of Exendin-4 on Liver and Kidney Lipids

To determine whether GLP-1–induced enhanced PPAR-α expression in the kidney and liver results in decreased hepatic and renal lipid accumulation, we directly measured the content of lipids in the liver and kidney. Exendin-4 treatment lowered total cholesterol, triglyceride, and FFA levels in the liver in a dosage-dependent manner in agreement with the pathologic changes (Table 4, Figure 3). In contrast to the liver, there was no difference in total cholesterol levels in the kidney in db/db mice with or without exendin-4 treatment. However, exendin-4 treatment significantly decreased triglyceride and FFA levels in the kidney in 1.0 exendin-4 db/db mice (P < 0.05 compared with db/db control; Table 4).

Discussion

We evaluated the role of GLP-1 activation using the GLP-1 analog exendin-4 in the development and progression of diabetic nephropathy in db/db mice. Exendin-4 treatment improved metabolic anomalies, including decreasing the body weight and adipose tissue weight. Exendin-4 treatment also improved fatty liver with an accompanying decrease in circulating FFA and triglyceride concentrations and improved insulin sensitivity in a dosage-dependent manner. In contrast, no difference in blood glucose and HbA_1c concentrations was detected in db/db mice. Importantly, these studies provide the first evidence that exendin-4 treatment ameliorates the severity of diabetic nephropathy with decreased glomerular hyperfiltration, extracellular matrix formation, inflammation, and apoptosis. This was associated with an increase in renal PPAR-α expression and a decrease in lipid accumulation in the renal tissue and oxidative stress as determined by urinary 8-OH-dG concentrations.

The db/db mouse is characterized by obesity, sustained hyperglycemia, hyperlipidemia, and insulinemia as a result of a destroyed leptin receptor on the C57BLKS/J background. Renal changes are characterized by glomerular hypertrophy, thickening of the glomerular basement membrane, albuminuria, and mesangial matrix accumulation within 2 mo of onset of diabetes (^15,16). Exendin-4 delays the onset of diabetes in db/db mice (¹⁷) and decreases blood glucose in animals and humans with type 2 diabetes (^{8–10,18–20}). To date, the mechanisms for improved blood glucose levels after exendin-4 treatment in diabetic animals are not fully understood. In contrast, in this study, there was no improvement in blood glucose or HbA_1c concentrations in exendin-4–treated db/db mice. Nevertheless, exendin-4 treatment of db/db mice reduced weight gain, adipose tissue mass, circulating triglycerides, FFA, insulin concentrations, and HOMA_IR indices in a dosage-dependent manner. The reason for this difference in blood glucose and HbA_1c was unclear. This could be due to differences in food intake between this and previous studies in the experimental groups or differences in duration of the administration of exendin-4 (²¹). Despite the similar glucose levels in treated and untreated mice, we found that exendin-4 treatment improved intraperitoneal glucose tolerance in db/db mice. Therefore, we carefully infer from these results that prolonged exendin-4 treatment improves circulating triglyceride and FFA levels and insulin sensitivity.

Previous reports suggested that obesity is associated with renal insufficiency in animals and humans (^22,23). The pathophysiology of obesity-associated renal damage includes glomerular hyperfiltration and the presence of excess adipocytes, which produce nephropathic cytokines. Adipocytes release a variety of hormones and cytokines, including proinflammatory, such as IL-1, IL-6, TNF-α and leptin, and anti-inflammatory, such as adiponectin, which may act on the kidney. These cytokines also play a role in insulin resistance of obesity. In this study, exendin-4 treatment markedly decreased body weight and peri-epididymal fatty tissue in db/db mice without detectable changes in food intake. Furthermore, exendin-4 treatment reduced 24-h urine volume and creatinine clearance associated with decreased in water intake. These results are consistent with the possibility that the renoprotective effects of exendin-4 result from reduced obesity.

It is widely known that nonalcoholic steato-hepatitis is a consequence of perturbed hepatic fatty acid oxidation systems, leading to lipid storage in liver cells (²⁴). Recent studies demonstrated that exendin-4 treatment reverses hepatic steatosis in ob/ob mice by improving insulin sensitivity (²⁵). These studies also found that ob/ob mice exhibited reduced weight gain, serum glucose, and oxidative stress in the liver during exendin-4 treatment. GLP-1–treated hepatocytes resulted in increased cAMP production as well as reduced mRNA expression of stearyl-CoA desaturase 1 and other genes that are associated with fatty acid synthesis. In our study, we also showed that exendin-4 treatment nearly normalized hepatic steatosis in a dosage-dependent manner.

Lipid deposition in the kidney may not be a rare phenomenon in animal models and in humans (^26–29). Several investigators advocated that it may play a major role in renal damage, which is mediated by the peroxidized lipids. They demonstrated the co-localization of lipid deposits and increased TGF-β1 mRNA expression in animal models of human diseases, especially angiotensin II–induced animals (²¹). We also found abnormal lipid deposits in the renal tissue in db/db mice using oil red O staining and direct measurement of lipid contents. Treatment of db/db mice with exendin-4 significantly ameliorated lipid deposits in the kidney. Only a small fraction of lipid droplets were found in the glomerulus in db/db mice that were treated exendin-4, in agreement with a decrease in renal lipid contents. Conversely, no lipid depositions in the kidney were observed in db/m mice regardless of exendin-4 treatment.

We recently reported that activation of PPAR-α with fenofibrate ameliorated diabetes, insulin resistance, net weight gain, albuminuria, glomerular hypertrophy, and mesangial expansion in db/db mice (⁷). PPAR-α deficiency also exacerbated diabetic renal disease with increased albuminuria, glomerular sclerosis, and mesangial area expansion associated with increasing serum FFA and triglycerides. Furthermore, they exhibited increased macrophage infiltration and glomerular apoptosis. In vitro studies also demonstrated that high glucose increased the expression of type IV collagen, TGF-β1, and the number of leukocytes that were adherent to cultured mesangial cells (⁶). In this study, exendin-4 treatment increased renal PPAR-α expression in a dosage-dependent manner in both db/m and db/db mice. Consistent with our previous findings, this increase in PPAR-α expression was accompanied by reduced mesangial expansion, glomerular immunostaining for TGF-β1, type IV collagen, F4/80, and caspase-3. Furthermore, exendin-4 treatment significantly decreased 24-h urinary 8-OH-dG concentrations and glomerular immunostaining, consistent with a reduction in oxidative DNA damage and oxidative stress. A recent article demonstrated that PPAR-α ligands also inhibit H₂O₂-mediated activation of TGF-β1 in human mesangial cells (³⁰). These findings suggest that TGF-β1 expression mediated by oxidant stress may be suppressible by exendin-4–induced PPAR-α activation. Another interesting finding of this study is that the number of GLP-1R–positive cells was markedly decreased in the glomerulus in db/db mice compared with db/m mice. It is interesting that exendin-4 treatment increased GLP-1R–positive cells in the glomeruli of db/db and db/m mice in a dosage-dependent manner. These findings suggest that exendin-4 at least acts in a renoprotective role through increasing GLP-1R expression in the glomerulus in db/db mice.

Conclusion

Exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.

Disclosures

None.

Figure 1: Change in body weight (A) and weight gain (B) in diabetic db/db and nondiabetic db/m mice that were treated without or with 0.5 or 1.0 exendin-4 for 8 wk starting at age 8 wk. *P < 0.05, **P < 0.001 versus control db/db mice.

Figure 2: Changes in fasting blood glucose (A), glycated hemoglobin (HbA_1c; B), and intraperitoneal glucose tolerance test (C; after 8 wk of treatment) in diabetic db/db and nondiabetic db/m mice that were treated without or with 0.5 or 1.0 exendin-4 for 8 wk starting at age 8 wk. *P < 0.05, 1.0 exendin-4 versus control db/db mice; **P < 0.001, db/m mice with or without 1.0 exendin-4 versus control db/db mice.

Figure 3: Hepatic histopathology in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. Histopathology shows marked improvement of hepatic steatosis in db/db mice that received exendin-4 (B and C) compared with untreated db/db mice (A) in a dosage-dependent manner. A representative photomicrograph of liver in nondiabetic db/m mice without (D) and with (E) treatment with 1.0 mmol/kg exendin-4. Magnification, ×200 (hematoxylin and eosin stain).

Figure 4: Renal morphology and immunohistochemical staining for TGF-β, type IV collagen, and F4/80 in renal cortical glomeruli in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. A representative photomicrograph of mesangial matrix accumulation in periodic acid-Schiff (PAS)-stained diabetic db/db mice without (A) or with treatment with 0.5 or 1.0 nmol/kg exendin-4 (B and C, respectively) and nondiabetic db/m mice without (D) and with (E) treatment with 1.0 mmol/kg exendin-4. (F) Quantitative assessment of mesangial matrix fraction in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. Immunohistochemical staining for TGF-β, type IV collagen, and F4/80 in kidney cortical glomeruli. Representative immunostains for TGF-β (G through K), type IV collagen (M through Q), and F4/80 (S through W; F4/80-positive cells are depicted by arrowheads) in diabetic and nondiabetic peroxisome proliferator–activated receptor-α (PPAR-α) wild-type and knockout kidneys. Quantitative assessment of TGF-β (F), type IV collagen (L), and osteopontin (R) and F4/80 (Z) immunoreactivity in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. Statistical significance was calculated using Mann-Whitney test. *P < 0.05, **P < 0.01 versus control db/db mice. In F4/80, *P = 0.002, **P < 0.001. Magnification, ×400.

Figure 4: Renal morphology and immunohistochemical staining for TGF-β, type IV collagen, and F4/80 in renal cortical glomeruli in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. A representative photomicrograph of mesangial matrix accumulation in periodic acid-Schiff (PAS)-stained diabetic db/db mice without (A) or with treatment with 0.5 or 1.0 nmol/kg exendin-4 (B and C, respectively) and nondiabetic db/m mice without (D) and with (E) treatment with 1.0 mmol/kg exendin-4. (F) Quantitative assessment of mesangial matrix fraction in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. Immunohistochemical staining for TGF-β, type IV collagen, and F4/80 in kidney cortical glomeruli. Representative immunostains for TGF-β (G through K), type IV collagen (M through Q), and F4/80 (S through W; F4/80-positive cells are depicted by arrowheads) in diabetic and nondiabetic peroxisome proliferator–activated receptor-α (PPAR-α) wild-type and knockout kidneys. Quantitative assessment of TGF-β (F), type IV collagen (L), and osteopontin (R) and F4/80 (Z) immunoreactivity in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. Statistical significance was calculated using Mann-Whitney test. *P < 0.05, **P < 0.01 versus control db/db mice. In F4/80, *P = 0.002, **P < 0.001. Magnification, ×400.

Figure 5: Immunohistochemical staining for caspase-3–positive cells in cortical glomeruli. Immunoreactivity for caspase-3 and representative pictures (A through E) of expression in diabetic db/db (A through C) and nondiabetic db/m (D and E) cortical kidneys. Decreased caspase-3–positive cell expression was seen in 0.5 (B) or 1.0 (C) nmol/kg exendin-4–treated diabetic db/db mice. (F) Number of caspase-3–positive cells per glomerulus in diabetic db/db and nondiabetic db/m mice that were treated without (D) or with (E) exendin-4. Statistical significance was calculated using Mann-Whitney test. *P < 0.05, **P < 0.01 versus control db/db mice. Magnification, ×400.

Figure 6: Immunohistochemical expression of PPAR-α protein in renal cortical tubules in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. (A) Decreased and sparse staining for PPAR-α protein in diabetic control db/db mice. (B and C) Abundant PPAR-α protein localized to the nucleus and cytoplasm of the cortical tubules in diabetic db/db mice that were treated with exendin-4 in a dosage-dependent manner (0.5 or 1.0 nmol/kg exendin-4; B and C, respectively). (D and E) In nondiabetic db/m mice, exendin-4 increases PPAR-α protein expression in renal cortical tubules. (F) Quantitative assessment of PPAR-α immunoreactivity in renal cortical tubules in db/db and db/m mice that were treated without or with exendin-4. Statistical significance was calculated using Mann-Whitney test. *P < 0.05, **P < 0.01 versus control db/db mice. Magnification, ×400.

Figure 7: Oil red O staining. (A) Kidney section from control db/db mice. (B and C) Kidney sections form db/db mice that were treated with 0.5 or 1.0 exendin-4, respectively. (D and E) Kidney sections from db/m mice that were treated without (D) or with (E) exendin-4. Magnification, ×400.

Figure 8: (A) Administration of exendin-4 suppresses 24-h urinary 8-hydroxy-deoxyguanosine (8-OH-dG) concentrations in db/db and db/m mice. *P < 0.05, **P < 0.01 versus control db/db mice. Immunohistochemical expression of 8-OH-dG protein in renal glomeruli. In db/db mice, treatment with 1.0 nmol/kg exendin-4 markedly attenuates 8-OH-dG protein expression in renal glomeruli (dark brown nucleus) compared with control db/db mice (B and C, respectively). There was no difference in 8-OH-dG expression in db/m mice without or with exendin-4 treatment (D and E, respectively). Magnification, ×400.

Figure 9: Immunohistochemical expression of glucagon-like peptide-1 receptor (GLP-1R)-positive cells in the cortical glomeruli in diabetic db/db and nondiabetic db/m mice that were treated without or with exendin-4. Immunoreactivity for GLP-1R and representative pictures (A through E) in diabetic db/db (A through C) and nondiabetic db/m (D and E) in the cortical kidneys. Markedly increased GLP-1R–positive cells are seen in 0.5 (B) or 1.0 (C) nmol/kg exendin-4–treated diabetic db/db mice. In nondiabetic db/m mice, GLP-1R–positive cells are also markedly increased compared with db/db mice regardless of exendin-4 treatment (D and E, respectively). (F) Negative control. (G) Number of GLP-1R–positive cells per glomerulus in diabetic db/db and nondiabetic db/m that were treated without or with exendin-4. Statistical significance was calculated using Mann-Whitney test. *P < 0.001 versus db/db control; **P < 0.001 versus control 0.5 nmol/kg exendin db/db mice; #P < 0.004 versus 1.0 nmol/kg exendin db/db; ##P < 0.001 versus db/m control. Magnification, ×400.

Table 1: Influences of exendin-4 on metabolic and physiologic parameters in db/db and db/m micea

Table 2: Effects of exendin-4 on serum lipid profiles, insulin, and HOMA_IR in db/db and db/m micea

Table 3: Effects of exendin-4 on renal functional and structural parametersa

Table 4: Effects of exendin-4 on liver and kidney lipids

This work was supported by a grant of Baxter Korea from the Korean Society of Nephrology and by the National Institutes of Health (R01DK74116 to M.D.B.).