Long-Term Treatment of Glucagon-Like Peptide-1 Analog Exendin-4 Ameliorates Diabetic Nephropathy through Improving Metabolic Anomalies in db/db Mice
Cheol Whee Park*,
Hyeong Wook Kim*,
Seung Hyun Ko,
Ji Hee Lim*,
Gyeong Ryul Ryu,
Hyun Wha Chung*,
Sang Woo Han*,
Seog Jun Shin*,
Byung Kee Bang*,
Matthew D. Breyer and
Yoon Sik Chang*
* Divisions of Nephrology and Endocrinology and Metabolism, Department of Internal Medicine, Catholic University of Korea, Seoul, Korea; and Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, Tennessee
Address correspondence to: Dr. Yoon Sik Chang, Division of Nephrology, Department of Internal Medicine, The Catholic University of Korea, #62 Yoido-Dong, Youngdungpo-Ku, Seoul, Korea 150-713. Phone: +82-2-3779-1259; Fax: +82-2-786-7725; E-mail: ysc543{at}unitel.co.kr
Received for publication July 23, 2006.
Accepted for publication January 31, 2007.
Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone andis a new clinically available class of agents for improvingof insulin resistance in both animals and humans with type 2diabetes. These studies aimed to determine whether long-termtreatment with a long-acting GLP-1 analog, exendin-4, delayedthe progression of diabetes. Male db/db mice and db/m mice at8 wk of age were treated with exendin-4 for 8 wk, whereas thecontrol db/db mice received only vehicle. Urinary albumin excretionwas significantly decreased in db/db mice that were treatedwith 1 nmol/kg exendin-4 compared with those in db/db mice thatwere treated with 0.5 nmol/kg exendin-4 and control db/db mice(P < 0.005). Intraperitoneal glucose tolerance test was improvedin db/db mice that were treated with 1 nmol/kg exendin-4 comparedwith other groups (P < 0.05). Despite this, fasting bloodglucose, glycated hemoglobin, and creatinine concentrationswere not significantly different among db/db mice. Renal histologystudies further demonstrated that glomerular hypertrophy, mesangialmatrix expansion, TGF-1 expression, and type IV collagen accumulationand associated glomerular lipid accumulation were significantlydecreased in db/db mice that were treated with 1 nmol/kg exendin-4.Furthermore, there were fewer infiltrating inflammatory cellsand apoptotic cells in the glomeruli of db/db mice that weretreated with 1 nmol/kg exendin-4 compared with those in theother groups accompanied by an increase in the renal immunoreactivityof peroxisome proliferator–activated receptor and GLP-1receptor–positive cells and a decrease in 24-h urinary8-hydroxy-deoxyguanosine levels (P < 0.01, respectively)along with decreases in lipid content. Taken together, exendin-4treatment seems to ameliorate diabetic nephropathy togetherwith improvement of the metabolic anomalies. These results suggestthat exendin-4 could provide a therapeutic role in diabeticnephropathy that results from type 2 diabetes.
In addition to the prominent role played by hyperglycemia, hypertension,growth factors (including angiotensin II, TGF-, connective tissuegrowth factor, and vascular endothelial growth factor), inflammatorycytokines, oxidative stress, and advanced glycation end products,abnormal lipid metabolism as well as renal accumulation of lipidshave been proposed to play a role in the pathogenesis of diabeticnephropathy (1,2). A recent study demonstrated that in diabetes,hyperglycemia per se directly upregulates renal expression ofthe transcriptional factor sterol regulatory element-bindingprotein-1, which caused increased fatty acid synthesis and accumulationof triglycerides. This was associated with upregulation of TGF-and vascular endothelial growth factor expression and accumulationof collagen and fibronectin, which result in mesangial expansion,glomerulosclerosis, and proteinuria (3).
Accumulation of excess lipids in nonadipose tissues leads tocell dysfunction or cell death (4,5). This phenomenon, knownas lipotoxicity, may play an important role in the pathogenesisof diabetes and heart failure in humans (4). Several mechanismshave been demonstrated to underlie lipotoxicity, including adirect 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 previousstudies demonstrated that the activation of peroxisome proliferator–activatedreceptor- (PPAR-) by fenofibrate improves diabetic nephropathyin db/db mice, and the lack of the PPAR- accelerates diabeticnephropathy in streptozotocin-induced type 1 diabetes (6,7).
Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone andalso considered a potential therapeutic agent for type 2 diabetesbecause it stimulates cell proliferation and insulin secretionin a glucose-dependent manner, inhibits glucagon secretion,induces satiety, and delays gastric emptying, which togetherresult in reduced circulating glucose (8,9). However, this peptideis almost immediately degraded by dipeptidyl peptidase-IV. Ina clinical study, a group of patients who had type 2 diabetesand received GLP-1 as a continuous subcutaneous infusion for6 wk showed that fasting and average plasma glucose concentrationswere lowered by approximately 5 mmol/L and free fatty acids(FFA) were significantly lowered, and the patients lost 2 kgin weight (10). Furthermore, insulin sensitivity and insulinsecretion capacity greatly improved. Despite the marked metabolicimprovement, plasma glucose levels were not completely normalized.
Exendin-4 is a 39–amino acid peptide that originally wasisolated from the salivary secretions of the Gila monster lizard(11). It shares approximately 53% homology with the mammalianincretin GLP-1 and binds to and activates the mammalian receptorfor GLP-1 cloned from pancreatic cells (12,13). These studiesexamined the effect of GLP-1, using the long-acting GLP-1 analogexendin-4, on the development and progression of diabetic nephropathyin db/db mice.
Experimental Methods
All experiments were performed according to institutional animalcare guidelines. Six-week-old male C57BLKS/J db/db and db/mmice were purchased from Jackson Laboratories (Bar Harbor, ME);db/m mice were used as controls in all experiments. Mice weremaintained on a 12-h light/dark cycle and were fed a standardlaboratory diet and water ad libitum in a room that was controlledfor temperature (23 ± 3°C) and humidity (55 ±15%). Exendin-4 (Sigma, St. Louis, MO) in 0.5 or 1.0 nmol/kgper d (0.5 or 1.0 exendin-4, respectively) was administratedintraperitoneally to db/db mice (n = 8, respectively) and age-and gender-matched db/m mice (n = 7) for 8 wk starting at ageof 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. Mousebody weight was measured weekly. Blood glucose was measuredevery 2 wk, and glycated hemoglobin (HbA1c) and 24-h urinaryalbumin and creatinine were measured every 4 wk. At 8 wk, intraperitonealglucose tolerance and systolic BP were assessed by a noninvasivetail-cuff system in conscious mice at the end of the study (IITCLife Science, Woodland Hills, CA). Mice were habituated to thetail-cuff device before measurement of the BP for 5 d over 15min to reduce variability of the measurements. After 8 wk, micewere anesthetized by an intraperitoneal injection of pentobarbitalsodium (55 mg/kg body wt; Nembutal; Boehringer Ingelheim, Artarmon,NSW, Australia). The kidneys were rapidly dissected and storedin buffered formalin (10%) for subsequent immunohistochemicalanalyses.
Measurement of Serum Parameters
Blood was collected from the left ventricle and centrifuged,and plasma was stored at –70°C for subsequent analyses.HbA1c was determined on red cell lysates by HPLC (BioRad, Richmond,CA). Total cholesterol, triglyceride, FFA, and insulin concentrationswere measured by autoanalyzer (Wako, Osaka, Japan). Homeostasismodel assessment for insulin resistance (HOMAIR) index was calculatedas follows: Fasting glucose (mmol/L) x fasting insulin (mU/L)/22.5.
Assessment of Renal Function
At weeks 0, 4, and 8, the mice were housed in metabolic cagesfor 24 h to collect urine for subsequent measurements of albuminconcentration by an immunoassay (Bayer, Elkhart, IN). At week8, plasma and urinary creatinine and urea concentrations weremeasured using HPLC and autoanalyzer (Beckman Instruments, Fullerton,CA), respectively. Creatinine clearance was calculated by (urine[Cr] x urine volume)/(plasma [Cr] x time).
Light Microscopic Study
Kidney and liver samples were collected after systemic perfusionwith PBS and then fixed in 4% paraformaldehyde. Histology wasassessed after hematoxylin and eosin and periodic acid-Schiffstaining. To examine the effect of exendin-4 on glomerular areaand mesangial matrix area, we performed glomerular analysison periodic acid-Schiff–stained kidney sections. Mesangialmatrix area and glomerular tuft area were quantified for eachglomerular cross-section as previously reported (6,7). Morethan 30 glomeruli that were cut through the vascular pole werecounted per kidney, and the average was used for analysis. Furthermore,to evaluate the effect of exendin-4 on lipid accumulation inthe glomerulus, we performed oil red O on staining frozen renaltissue.
Immunohistochemistry for TGF-1, Type IV Collagen, F4/80, Caspase-3, PPAR-, GLP-1R, and 8-Hydroxy-Deoxyguanosine
We performed immunohistochemistry for type IV collagen, TGF-1,F4/80, caspase-3, PPAR-, 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 withPBS, and incubated with 1.5% H2O2 in methanol to block endogenousperoxidase activity. Nonspecific binding was blocked with 10%normal goat serum in PBS. Sections were incubated overnightwith the anti–type IV collagen (1:150 in blocking solution;Biodesign Int., Saco, ME), anti–TGF-1 (1:100; Santa CruzBiotechnology, Santa Cruz, CA), anti-F4/80 (1:100; Serotek,Oxford, UK), anti–caspase-3 (1:50; Santa Cruz Biotechnology),anti–PPAR- (1:1500; provided by Dr. A. Sugihara, TokohuUniversity 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. Tissuesections were treated with an antigen-unmasking solution thatconsisted of 10 mM Na citrate (pH 6.0) and 0.05% Tween 20. Antibodieswere localized with the ABC technique (Vector Laboratories,Burlingame, CA) and 3,3-diaminobenzidine substrate solutionwith nickel chloride enhancement. Sections were then dehydratedin ethanol, cleared in xylene, and mounted without counterstaining.
All of these sections were examined in a masked manner usinglight microscopy (Olympus BX-50; Olympus Optical, Tokyo, Japan).For the quantification of proportional area of staining, approximately20 views (x400 magnification) were randomly located in the renalcortex and corticomedullary junction of each slide (Scion ImageBeta 4.0.2, Frederick, MD).
24-Hour Urinary 8-OH-dG
To determine the oxidative DNA damage in the kidney, we determined24-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 kitsfrom Waco Co., (Osaka, Japan). Liver and kidney lipids wereextracted by the method of Bligh and Dyer with slight modifications(14). A portion (50 mg) of the liver or kidney was homogenizedand extracted by methanol-chloroform aliquots (2:1) in a 37°Cwater 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 betweenthe groups were examined for statistical significance usingANOVA with Bonferroni correction (SPSS 11.5, Chicago, IL). P< 0.05 was considered a statistically significant difference.
Body Weight, Liver Weight, Peri-Epididymal Fatty Tissue Weight, and the Levels of Glucose and HbA1c
Body weight of db/db mice that were treated with 0.5 or 1.0exendin-4 was less than that of control db/db mice at the endof experimentation (P < 0.05; Figure 1A). There was no suchdifference between db/m mice that were treated with or withoutexendin-4. There was significant decrease in body weights indb/db mice that were treated with 0.5 or 1.0 exendin-4 (P <0.05, respectively; Figure 1B) compared with those of controldb/db mice. The weight of peri-epididymal fat of db/db micethat were treated with 0.5 or 1.0 exendin-4 was significantlydecreased compared with control db/db mice (P < 0.01 andP < 0.001, respectively; Table 1). There was no differencein liver weight and food intake among db/db mice that were treatedwith or without exendin-4 (Table 1). Diabetic db/db mice exhibiteddramatically increased blood glucose and HbA1c levels throughoutthe entire period of the experiment (P < 0.001; Figure 2)compared with those of nondiabetic db/m mice. However, therewere no significant differences in blood glucose and HbA1c amongdb/db mice.
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 (HbA1c; 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.
Blood Chemistry, Serum Lipid and Insulin Levels, and HOMAIR Index
There was no difference in hematocrit among diabetic db/db micewith or without exendin-4 treatment (Table 1). In contrast,serum FFA and triglyceride levels were significantly decreasedin db/db mice that were treated with 1.0 exendin-4 comparedwith those of control db/db mice (P < 0.05; Table 2). Furthermore,serum insulin concentrations (P = 0.05 and P = 0.001, respectively)and HOMAIR indices (P = 0.011 and P < 0.001, respectively)were markedly decreased in db/db mice that were treated withexendin-4 in a dosage-dependent manner (Table 2). There wasno such difference between db/m mice that were treated withor without exendin-4. It is interesting that there were no differencesin total cholesterol concentrations among the groups (Table 2).
Table 2. Effects of exendin-4 on serum lipid profiles, insulin, and HOMAIR in db/db and db/m micea
Kidney Weight, Albuminuria, Serum Creatinine, Creatinine Clearance, and Systolic BP
The kidney weight, blood urea nitrogen, and creatinine in blooddid not differ significantly between experimental groups (Table 3).As expected, db/db mice exhibited an increase in 24-h urinevolumes; 1.0 exendin-4 treatment in db/db mice profoundly decreasedurine volume (P < 0.01; Table 3), resulting from decreasedwater intake (P = 0.03; Table 1). Diabetic db/db mice also exhibiteda persistent increase in creatinine clearance and urine albuminexcretion, whereas creatinine clearance by HPLC and albuminuriain exendin-4–treated db/db mice decreased to the levelsof db/m mice in a dosage-dependent manner (P < 0.05 and P= 0.005, respectively; Table 3). No significant differencesin systolic BP (Table 1) were observed among the groups.
Table 3. Effects of exendin-4 on renal functional and structural parametersa
Liver and Renal Histologic Examination
In db/db mice, severe macrovesicular fatty liver was prominentin the whole liver; however, exendin-4 treatment markedly improvedhepatic steatosis in a dosage-dependent manner (Figure 3). Glomerularinjury in diabetic db/db mice was characterized by mesangialextracellular matrix expansion and glomerulosclerosis (Figure 4,A through E). Increased glomerular fractional mesangial areaswere decreased in db/db mice that were treated with 1.0 exendin-4(P < 0.01; Figure 4F), but there was no difference betweenglomerular fractional mesangial ares in db/db mice and thoseof 0.5 exendin-4–treated 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, x200 (hematoxylin and eosin stain).
Figure 4. Renal morphology and immunohistochemical staining for TGF-, 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-, type IV collagen, and F4/80 in kidney cortical glomeruli. Representative immunostains for TGF- (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- (PPAR-) wild-type and knockout kidneys. Quantitative assessment of TGF- (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, x400.
Expression of TGF-1, Type IV Collagen, and Macrophage Infiltration
Diabetes was associated with an increase in TGF-1 expressionin the glomerulus (Figure 4G). In the kidneys of exendin-4–treateddb/db mice, positive staining for TGF-1 was significantly decreasedand localized to the glomerulus in a dosage-dependent manner(P < 0.05 and P < 0.01, respectively; Figure 4, H andI). In contrast, there was no such change in db/m mice (Figure 4,J, K, and L). Diabetes was also associated with an increasein type IV collagen protein expression in the glomerulus (Figure 4,M through Q). The prominent increase in type IV collagen inthe glomerulus in control db/db mice was markedly decreasedin exendin-4–treated db/db mice in a dosage-dependentmanner (P < 0.05 and P < 0.01; Figure 4R).
Only modest macrophage infiltration, as assessed by F4/80-positivestaining, was observed in the glomerulus of db/m mice with orwithout exendin-4 treatment (Figure 4, S through W). In contrast,F4/80 immunostaining was markedly increased in the glomerulusof db/db mice compared with that of db/m mice (Figure 4S). Exendin-4treatment significantly decreased the expression of F4/80 immunostainingin 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–positivecells were observed within the glomeruli (Figure 5, A throughE). In db/db mice, diabetes was associated with a markedly increasednumber of glomerular caspase-3–positive cells comparedwith those of other db/db mice groups (P < 0.001; Figure 5A).In contrast, when db/db mice were treated with exendin-4, especially1.0 exendin-4, caspase-3 immunostaining was markedly decreased(P < 0.001; Figure 5, E and F).
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, x400.
Expression of PPAR- and Lipid Staining in the Glomerulus
Immunohistochemistry revealed significantly increased PPAR-expression in exendin-4–treated db/db kidney comparedwith that in control db/db kidneys (Figure 6). The increasesin renal PPAR- immunostaining in the db/db mice that were treatedwith 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 proximaltubules and thick ascending tubules (Figure 6, B and C). Oilred O staining of kidney sections showed no apparent lipid depositionin the kidneys of control db/m mice regardless of the treatmentof exendin-4 (Figure 7, D and E). Conversely, in db/db mice,marked accumulation of oil red O–stainable lipid was observedin the glomerular and tubular epithelial cells in kidney section(Figure 7A). Only a small fraction of lipid droplets were foundin the glomerulus in db/db mice that were treated with both0.5 and 1.0 exendin-4, respectively (Figure 7, B and C).
Figure 6. Immunohistochemical expression of PPAR- 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- protein in diabetic control db/db mice. (B and C) Abundant PPAR- 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- protein expression in renal cortical tubules. (F) Quantitative assessment of PPAR- 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, x400.
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, x400.
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 urinary8-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, weperformed immunostaining of 8-OH-dG expression in the kidney.In the glomerulus, strongly increased immunostaining expressionof 8-OH-dG (dark brown nucleus) in db/db mice markedly attenuatedby treatment with 1.0 exendin-4 (Figure 8C) compared with controldb/db mice (Figure 8B). However, there was no difference in8-OH-dG immunostaining in db/m mice with or without exendin-4treatment (Figure 8, D and E). To determine the effect of exendin-4on GLP-1R expression in the glomerulus, we also performed immunostainingfor GLP-1R in the kidney. Noticeably increased GLP-1R–positivecell expression was seen in the glomeruli of db/db mice thatwere 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 with0.5 exendin-4–treated db/db mice; Figure 9C). In db/mmice, the expressions of GLP-1R–positive cells was evidentlyincreased in db/m compared with exendin-4–treated db/dbmice (P < 0004 compared with 1.0 exendin-4 db/db [Figure 9D];P < 0.001 compared with db/m control [Figure 9E]).
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, x400.
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, x400.
Effect of Exendin-4 on Liver and Kidney Lipids
To determine whether GLP-1–induced enhanced PPAR- expressionin the kidney and liver results in decreased hepatic and renallipid accumulation, we directly measured the content of lipidsin the liver and kidney. Exendin-4 treatment lowered total cholesterol,triglyceride, and FFA levels in the liver in a dosage-dependentmanner in agreement with the pathologic changes (Table 4, Figure 3).In contrast to the liver, there was no difference in total cholesterollevels in the kidney in db/db mice with or without exendin-4treatment. However, exendin-4 treatment significantly decreasedtriglyceride and FFA levels in the kidney in 1.0 exendin-4 db/dbmice (P < 0.05 compared with db/db control; Table 4).
We evaluated the role of GLP-1 activation using the GLP-1 analogexendin-4 in the development and progression of diabetic nephropathyin 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 accompanyingdecrease in circulating FFA and triglyceride concentrationsand improved insulin sensitivity in a dosage-dependent manner.In contrast, no difference in blood glucose and HbA1c concentrationswas detected in db/db mice. Importantly, these studies providethe first evidence that exendin-4 treatment ameliorates theseverity of diabetic nephropathy with decreased glomerular hyperfiltration,extracellular matrix formation, inflammation, and apoptosis.This was associated with an increase in renal PPAR- expressionand a decrease in lipid accumulation in the renal tissue andoxidative 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 leptinreceptor on the C57BLKS/J background. Renal changes are characterizedby glomerular hypertrophy, thickening of the glomerular basementmembrane, albuminuria, and mesangial matrix accumulation within2 mo of onset of diabetes (15,16). Exendin-4 delays the onsetof diabetes in db/db mice (17) and decreases blood glucose inanimals and humans with type 2 diabetes (8–10,18–20).To date, the mechanisms for improved blood glucose levels afterexendin-4 treatment in diabetic animals are not fully understood.In contrast, in this study, there was no improvement in bloodglucose or HbA1c concentrations in exendin-4–treated db/dbmice. Nevertheless, exendin-4 treatment of db/db mice reducedweight gain, adipose tissue mass, circulating triglycerides,FFA, insulin concentrations, and HOMAIR indices in a dosage-dependentmanner. The reason for this difference in blood glucose andHbA1c was unclear. This could be due to differences in foodintake between this and previous studies in the experimentalgroups or differences in duration of the administration of exendin-4(21). Despite the similar glucose levels in treated and untreatedmice, we found that exendin-4 treatment improved intraperitonealglucose tolerance in db/db mice. Therefore, we carefully inferfrom these results that prolonged exendin-4 treatment improvescirculating triglyceride and FFA levels and insulin sensitivity.
Previous reports suggested that obesity is associated with renalinsufficiency in animals and humans (22,23). The pathophysiologyof obesity-associated renal damage includes glomerular hyperfiltrationand the presence of excess adipocytes, which produce nephropathiccytokines. Adipocytes release a variety of hormones and cytokines,including proinflammatory, such as IL-1, IL-6, TNF- and leptin,and anti-inflammatory, such as adiponectin, which may act onthe kidney. These cytokines also play a role in insulin resistanceof obesity. In this study, exendin-4 treatment markedly decreasedbody weight and peri-epididymal fatty tissue in db/db mice withoutdetectable changes in food intake. Furthermore, exendin-4 treatmentreduced 24-h urine volume and creatinine clearance associatedwith decreased in water intake. These results are consistentwith the possibility that the renoprotective effects of exendin-4result from reduced obesity.
It is widely known that nonalcoholic steato-hepatitis is a consequenceof perturbed hepatic fatty acid oxidation systems, leading tolipid storage in liver cells (24). Recent studies demonstratedthat exendin-4 treatment reverses hepatic steatosis in ob/obmice by improving insulin sensitivity (25). These studies alsofound 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 productionas well as reduced mRNA expression of stearyl-CoA desaturase1 and other genes that are associated with fatty acid synthesis.In our study, we also showed that exendin-4 treatment nearlynormalized hepatic steatosis in a dosage-dependent manner.
Lipid deposition in the kidney may not be a rare phenomenonin animal models and in humans (26–29). Several investigatorsadvocated that it may play a major role in renal damage, whichis mediated by the peroxidized lipids. They demonstrated theco-localization of lipid deposits and increased TGF-1 mRNA expressionin animal models of human diseases, especially angiotensin II–inducedanimals (21). We also found abnormal lipid deposits in the renaltissue in db/db mice using oil red O staining and direct measurementof lipid contents. Treatment of db/db mice with exendin-4 significantlyameliorated lipid deposits in the kidney. Only a small fractionof lipid droplets were found in the glomerulus in db/db micethat were treated exendin-4, in agreement with a decrease inrenal lipid contents. Conversely, no lipid depositions in thekidney were observed in db/m mice regardless of exendin-4 treatment.
We recently reported that activation of PPAR- with fenofibrateameliorated diabetes, insulin resistance, net weight gain, albuminuria,glomerular hypertrophy, and mesangial expansion in db/db mice(7). PPAR- deficiency also exacerbated diabetic renal diseasewith increased albuminuria, glomerular sclerosis, and mesangialarea expansion associated with increasing serum FFA and triglycerides.Furthermore, they exhibited increased macrophage infiltrationand glomerular apoptosis. In vitro studies also demonstratedthat high glucose increased the expression of type IV collagen,TGF-1, and the number of leukocytes that were adherent to culturedmesangial cells (6). In this study, exendin-4 treatment increasedrenal PPAR- expression in a dosage-dependent manner in bothdb/m and db/db mice. Consistent with our previous findings,this increase in PPAR- expression was accompanied by reducedmesangial expansion, glomerular immunostaining for TGF-1, typeIV collagen, F4/80, and caspase-3. Furthermore, exendin-4 treatmentsignificantly decreased 24-h urinary 8-OH-dG concentrationsand glomerular immunostaining, consistent with a reduction inoxidative DNA damage and oxidative stress. A recent articledemonstrated that PPAR- ligands also inhibit H2O2-mediated activationof TGF-1 in human mesangial cells (30). These findings suggestthat TGF-1 expression mediated by oxidant stress may be suppressibleby exendin-4–induced PPAR- activation. Another interestingfinding of this study is that the number of GLP-1R–positivecells was markedly decreased in the glomerulus in db/db micecompared with db/m mice. It is interesting that exendin-4 treatmentincreased GLP-1R–positive cells in the glomeruli of db/dband db/m mice in a dosage-dependent manner. These findings suggestthat exendin-4 at least acts in a renoprotective role throughincreasing GLP-1R expression in the glomerulus in db/db mice.
Exendin-4 treatment seems to ameliorate diabetic nephropathytogether with improvement of metabolic anomalies. These resultssuggest that exendin-4 could provide a therapeutic role in diabeticnephropathy that results from type 2 diabetes.
This work was supported by a grant of Baxter Korea from theKorean Society of Nephrology and by the National Institutesof Health (R01DK74116 to M.D.B.).
Footnotes
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Top
Abstract
Introduction
1 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 
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. 
