Ji Hee Lim

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

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-beta1 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 alpha 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.

Peroxisome proliferator-activated receptor-α activator fenofibrate prevents high-fat diet-induced renal lipotoxicity in spontaneously hypertensive rats

We investigated the effects of a high-fat (HF) diet and peroxisome proliferator-activated receptor (PPAR)-α activation on the intrarenal lipotoxicity associated with the renin–angiotensin system (RAS) and oxidative stress using spontaneously hypertensive (SHR) rats. Male SHR and Wistar–Kyoto (WKY) rats at 8 weeks of age were fed either a normal-fat diet or an HF diet without or with fenofibrate treatment for 12 weeks. Severe intrarenal lipid accumulation was noted in the SHR rats fed an HF diet than in WYK rats fed an HF diet (P<0.05). This lipid accumulation was associated with a 70% decrease in renal PPARα expression in SHR rats, whereas an HF diet increased the expression of PPARα in WKY rats by threefold. An HF diet also activated intrarenal, not systemic, RAS and induced oxidative stress associated with reduced nitric oxide (NO) bioavailability. By contrast, fenofibrate attenuated weight gain, fat mass and insulin resistance. Fenofibrate recovered HF diet-induced decreases in intrarenal PPARα expression and fat accumulation, and abolished intrarenal RAS activation and oxidative stress in SHR–HF animals (P<0.01). These activities conferred protection against increased blood pressure (BP), glomerulosclerosis and renal inflammation. Intrarenal free fatty acid and triglyceride concentrations were positively correlated with angiotensin II (γ=0.63, 0.36) and 24-h urinary 8-hydroxy-deoxyguanosine (γ=0.36, 0.39), and negatively correlated with PPARα contents (γ=−0.47, −0.44; P<0.05). An HF diet-induced lipotoxicity by depletion of intrarenal PPARα aggravated BP and renal inflammation as a result of intrarenal RAS activation and oxidative stress. Therefore, intervention with PPARα activators can effectively prevent diet-induced renal lipotoxicity in hypertensive rats.

Fenofibrate Improves Renal Lipotoxicity through Activation of AMPK-PGC-1α in db/db Mice

Peroxisome proliferator-activated receptor (PPAR)-α, a lipid-sensing transcriptional factor, serves an important role in lipotoxicity. We evaluated whether fenofibrate has a renoprotective effect by ameliorating lipotoxicity in the kidney. Eight-week-old male C57BLKS/J db/m control and db/db mice, divided into four groups, received fenofibrate for 12 weeks. In db/db mice, fenofibrate ameliorated albuminuria, mesangial area expansion and inflammatory cell infiltration. Fenofibrate inhibited accumulation of intra-renal free fatty acids and triglycerides related to increases in PPARα expression, phosphorylation of AMP-activated protein kinase (AMPK), and activation of Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α)-estrogen-related receptor (ERR)-1α-phosphorylated acetyl-CoA carboxylase (pACC), and suppression of sterol regulatory element-binding protein (SREBP)-1 and carbohydrate regulatory element-binding protein (ChREBP)-1, key downstream effectors of lipid metabolism. Fenofibrate decreased the activity of phosphatidylinositol-3 kinase (PI3K)-Akt phosphorylation and FoxO3a phosphorylation in kidneys, increasing the B cell leukaemia/lymphoma 2 (BCL-2)/BCL-2-associated X protein (BAX) ratio and superoxide dismutase (SOD) 1 levels. Consequently, fenofibrate recovered from renal apoptosis and oxidative stress, as reflected by 24 hr urinary 8-isoprostane. In cultured mesangial cells, fenofibrate prevented high glucose-induced apoptosis and oxidative stress through phosphorylation of AMPK, activation of PGC-1α-ERR-1α, and suppression of SREBP-1 and ChREBP-1. Our results suggest that fenofibrate improves lipotoxicity via activation of AMPK-PGC-1α-ERR-1α-FoxO3a signaling, showing its potential as a therapeutic modality for diabetic nephropathy.

Age-Associated Molecular Changes in the Kidney in Aged Mice

Background. Aging is a multifactorial process characterized by a progressive decline in physiological function. Decreased kidney function is associated with cardiovascular disease and mortality. Therefore, increasing our insight into kidney aging by understanding the anatomic, physiologic, and pathologic changes of aging in the kidney is important to prevent disastrous outcomes in elderly people. Methods. Male two-, 12-, and 24-month-old C57/BL6 mice were used in this study. We measured histological change, oxidative stress, and aging-related protein expression in the kidneys. Results. Twenty-four-month-old mice displayed increased albuminuria. Creatinine clearance decreased with aging, although this was not statistically significant. There were increases in mesangial volume and tubulointerstitial fibrosis in 24-month-old mice. There were also increases in F4/80 expression and in apoptosis detected by TUNEL assay. Urine isoprostane excretion increased with aging and SOD1 and SOD2 were decreased in 24-month-old mice. Oxidative stress may be mediated by a decrease in Sirt1, PGC-1α, ERR-1α, and PPARα expression. Klotho expression also decreased. Conclusions. Our results demonstrate that Sirt1 was decreased with aging and may relate to changed target molecules including PGC-1α/ERR-1α signaling and PPARα. Klotho can also induce oxidative stress. Pharmacologically targeting these signaling molecules may reduce the pathologic changes of aging in the kidney.

High-fat diet-induced renal cell apoptosis and oxidative stress in spontaneously hypertensive rat are ameliorated by fenofibrate through the PPARα–FoxO3a–PGC-1α pathway

BACKGROUND The peroxisome proliferator-activated receptor-α (PPARα) is a lipid-sensing transcriptional factor that has a role in gluco-oxidative stress and lipotoxicity. Forkhead box O (FoxO)s and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α are also known to regulate cell metabolism, cell cycle arrest, apoptosis and oxidative stress during stressful conditions. We evaluated whether PPARα-FoxOs-PGC-1α signaling in overfed spontaneously hypertensive rats (SHR) has a protective role in the kidney. METHODS Male SHR and Wistar-Kyoto rats (WKY) fed a high-fat diet (HFD) received treatment with fenofibrate, PPARα agonist or tempol, antioxidants for 12 weeks and were evaluated about the PPARα-FoxOs-PGC-1α pathway. RESULTS The SHRs with an HFD had an elevated systolic pressure, plasma insulin, free fatty acid (FFA) and triglyceride (TGs) levels, and they had induced glucose intolerance as well as albuminuria, glomerular expansion and renal inflammation. An HFD caused the accumulation of intra-renal FFA and TGs and this was related to a decrease in the PPARα expression, the activation of phosphatidylinositol 3-kinase (PI3K)-Akt, phosphorylation of FoxO3a and decreases in the PGC-1α and estrogen-related receptor (ERR)-1α expressions, which suppressed the superoxide dismutase (SOD2) and Bcl-2 expressions and led to increases in oxidative stress and the number of apoptotic renal cells. Interestingly, administering fenofibrate or tempol to the HFD-induced SHRs reversed all of the renal phenotypes by increasing the PPARα expression with concomitant inactivation of the PI3K-Akt pathway, dephosphorylation of FoxO3a and activation of PGC-1α-ERR-1α signaling, and this all resulted in ameliorating the oxidative stress and apoptotic cell death. CONCLUSION Our results demonstrated that PPARα agonists or antioxidants are associated with improvement of the circulating FFA and TGs levels and this prevents HFD-induced renal lipotoxicity and hypertension by the activation of PPARα and its downstream signals of both FoxO3a and PGC-1α.

Tempol or candesartan prevents high-fat diet-induced hypertension and renal damage in spontaneously hypertensive rats

BACKGROUND Obesity has been strongly associated with the development and aggravation of hypertension and chronic kidney disease. To date, the systemic renin-angiotensin system (RAS) has been known to involve in obesity-induced tissue damage and hypertension. However, the intrarenal mechanism whereby obesity induces and aggravates hypertension and renal disease remains poorly understood. Therefore, we investigated the role of intrarenal RAS and oxidative stress in diet-induced hypertension and renal inflammation in spontaneously hypertensive rats (SHR) fed a high-fat diet. METHODS Male SHR and Wistar-Kyoto rats (WKY) were divided into eight groups: normal-fat diet-fed WKY (WKY-NF), high-fat diet-fed WKY (WKY-HF), high-fat diet-fed tempol-treated WKY (WKY-HF/T), high-fat diet-fed candesartan-treated WKY (WKY-HF/C), normal-fat diet-fed SHR (SHR-NF), high-fat diet-fed SHR (SHR-HF), high-fat diet-fed tempol-treated SHR (SHR-HF/T) and high-fat diet-fed candesartan-treated SHR (SHR-HF/C). After 12 weeks of treatment, haemodynamic measurements and histological assessment of the kidney were performed. RESULTS At the end of week 12, the high-fat fed SHR gained more body weight, their systolic blood pressure was further elevated and glucose intolerance induced. There was no significant difference in the insulin resistance index, serum lipid profile, plasma renin activity and serum aldosterone levels according to diet. However, the high-fat diet resulted in increases in immunohistochemical stains of renin and angiotensin II in the kidney. The real-time PCR also demonstrated significant increases in mRNA levels of renin, angiotensinogen and angiotensin-converting enzyme in the kidney, reflecting enhanced activation of the intrarenal RAS, which findings were also shown by Western blot analysis for renin and angiotensin II type 1 receptor. The expression of ED-1, osteopontin and TGF-beta1 in the renal cortex were prominently enhanced in the SHR-HF group with the increased intrarenal lipid concentrations and oxidative stress. Administration of tempol or candesartan in the high-fat diet-induced SHR inhibited the elevation of the systolic blood pressure, intrarenal lipid concentrations, oxidative stress and the degree of renal inflammation to the levels of, or more than, the SHR-NF with no differences in the body weight and periepididymal fat weight, compared to those in the SHR-HF group without such treatment. CONCLUSIONS Our study suggests that a high-fat diet induces fatty kidneys, aggravation of blood pressure and renal inflammation in the SHR. Blockade of oxidative stress by tempol or of RAS by candesartan ameliorates the increase in blood pressure and renal inflammation and improves intrarenal lipid accumulation. Therefore, antioxidants or angiotensin receptor blockers can prevent diet-induced hypertension and renal inflammation in hypertensive rats.

Vascular Endothelial Growth Factor Inhibition by dRK6 Causes Endothelial Apoptosis, Fibrosis, and Inflammation in the Heart via the Akt/eNOS Axis in db/db Mice

OBJECTIVE Vascular endothelial growth factor (VEGF), which is associated with the stimulation of angiogenesis and collateral vessel synthase, is one of the crucial factors involved in cardiac remodeling in type 2 diabetes. RESEARCH DESIGN AND METHODS We investigated VEGF inhibition by dRK6 on the heart in an animal model of type 2 diabetes. Male db/db and db/m mice either were treated with dRK6 starting at 7 weeks of age for 12 weeks (db/db-dRK6 and db/m-dRK6) or were untreated. RESULTS Cardiac dysfunction and hypertrophy were noted by echocardiogram and molecular markers in the db/db-dRK6 mice. The presence of diabetes significantly suppressed the expression of VEGF receptor (VEGFR)-1 and VEGFR-2, phospho-Akt, and phospho-endothelial nitric oxide synthase (eNOS) in the heart. In db/db-dRK6 mice, dRK6 completely inhibited VEGFR-2, phospho-Akt, and phospho-eNOS expression, whereas no effect on VEGFR-1 was observed. Cardiac fibrosis, microvascular scarcity associated with an increase in apoptotic endothelial cells, and inflammation were prominent, as well as increase in antiangiogenic growth factors. Cardiac 8-hydroxy-deoxyguanine and hypoxia-inducible factor-1α expression were significantly increased. No such changes were found in the other groups, including the db/m-dRK6 mice. The number of apoptotic human umbilical vein endothelial cells was increased by dRK6 in a dose-dependent manner only at high glucose concentrations, and this was associated with a decrease in phospho-Akt and phospho-eNOS related to oxidative stress. CONCLUSIONS Our results demonstrated that systemic blockade of VEGF by dRK6 had deleterious effects on the heart in an animal model of type 2 diabetes; dRK6 induced downregulation of the VEGFR-2 and Akt-eNOS axis and enhancement of oxidative stress.

Vascular endothelial growth factor-receptor 1 inhibition aggravates diabetic nephropathy through eNOS signaling pathway in db/db mice.

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice. While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling. In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.

Therapeutic Effects of Fenofibrate on Diabetic Peripheral Neuropathy by Improving Endothelial and Neural Survival in db/db Mice

Neural vascular insufficiency plays an important role in diabetic peripheral neuropathy (DPN). Peroxisome proliferative-activated receptor (PPAR)α has an endothelial protective effect related to activation of PPARγ coactivator (PGC)-1α and vascular endothelial growth factor (VEGF), but its role in DPN is unknown. We investigated whether fenofibrate would improve DPN associated with endothelial survival through AMPK-PGC-1α-eNOS pathway. Fenofibrate was given to db/db mice in combination with anti-flt-1 hexamer and anti-flk-1 heptamer (VEGFR inhibition) for 12 weeks. The db/db mice displayed sensory-motor impairment, nerve fibrosis and inflammation, increased apoptotic cells, disorganized myelin with axonal shrinkage and degeneration, fewer unmyelinated fibers, and endoneural vascular rarefaction in the sciatic nerve compared to db/m mice. These findings were exacerbated with VEGFR inhibition in db/db mice. Increased apoptotic cell death and endothelial dysfunction via inactivation of the PPARα-AMPK-PGC-1α pathway and their downstream PI3K-Akt-eNOS-NO pathway were noted in db/db mice, human umbilical vein endothelial cells (HUVECs) and human Schwann cells (HSCs) in high-glucose media. The effects were more prominent in response to VEGFR inhibition. In contrast, fenofibrate treatment ameliorated neural and endothelial damage by activating the PPARα-AMPK-PGC-1α-eNOS pathway in db/db mice, HUVECs and HSCs. Fenofibrate could be a promising therapy to prevent DPN by protecting endothelial cells through VEGF-independent activation of the PPARα-AMPK-PGC-1α-eNOS-NO pathway.

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.