Effects And Mechanisms Of Fenofibrate And Leptin On Renal And Aortic Injury In Hypertension

BackgroudHypertension is an important risk factor for the progression of glomerular and tubulointerstitial diseases to chronic renal failure. The progression of hypertensive renal disease displays several characteristics, including proteinuria, inflammatory cell recruitment, and accumulation of extracellular matrix (ECM) proteins in the interstitium. Oxidative stress has been shown to play an important role in the development of renal injury, because it can stimulate the expression of proinflammatory and profibrotic molecules in addition to direct toxic effects. Conversely, antioxidants can attenuate the development of kidney injury.Peroxisome proliferator-activated receptors (PPARs), which include three membersα,γ, andβ/δ, are ligand-activated transcription factors belonging to the nuclear receptor superfamily. On activation by their ligands, PPARs bind to specific PPAR response elements (PPREs) in the promoter region of their target genes. PPARa, which is highly expressed in kidney, liver, and heart, has been shown to take part in diverse physiological processes, including maintenance of lipid and glucose homeostasis. In addition, PPARa also exerts antioxidant and anti-inflammatory effects. Activation of PPARa inhibits angiotensinⅡ-induced activation of NADPH oxidase and suppressed ROS production in the vascular wall. Furthermore, a PPAR-responsive element (PPRE) has been identified in promoter regions of catalase and Cu/Zn-SOD genes, which are key enzymes that reduce ROS production. Also, PPARa plays crucial roles in the attenuation of inflammatory response in activated mesangial cells through its antagonizing effects on the NF-κB signaling pathway. Furthermore, PPARa-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARa is also a modulator of inflammation. These studies have identified PPARa as a naturally occurring regulator of oxidative stress and inflammation. However, little is known about the effect of PPARa on chronic hypertensive renal damage.Taken together, we hypothesized that PPARa agonist fenofibrate might inhibit hypertensive renal injury by regulating oxidative stress and inflammation. The results from this study indicate that fenofibrate is able to inhibit renal injury in SHRs without affecting blood pressure by ameliorating renal inflammation and TGF-β1 expression via inhibition of oxidative stress and MAPK activity.Methods1. Animal models and experimental design:Twenty-four 8-week-old male SHRs and ten 8-week-old male Wistar-Kyoto (WKY) rats were used in this study. SHRs were divided into the following two groups:1) SHRs given normal oral 0.9% saline (n=10); 2) SHRs given oral fenofibrate at a dose of 60 mg·kg-1·d-1 dissolved in 0.9% saline (n=14). The WKY rats (n=10) given 0.9% saline were used as controls. The compound or vehicle was orally administered in 2 ml-kg"1 once a day for 18 weeks.2. Assessment of body weight and systolic blood pressure:Body weight was determined every two weeks throughout the study. Systolic blood pressure (SBP) was assessed every two weeks throughout the study using a tail-cuff method in conscious rats after prewarming at 38℃for 10 min.3. Assessment of UAE, BUN and Creatinine:At the end of the study, the animals were housed in metabolic cages for 24 h to collect urine for subsequent measurements of urinary protein excretion by an immunoassay, and then were anesthetized with pentobarbital, blood samples were collected from the right ventricle and serum was stored at -80℃for subsequently analyzing creatinine and blood urea nitrogen concentrations. 4. Histopathological study:Coronal sections of renal tissue (3 to 4-μm-thick) were stained with periodic acid-Schiff (PAS) and Masson trichrome for analysis of glomerular sclerosis and tubulointerstitial fibrosis, and examined by light microscopy in a blinded fashion. A semiquantitative morphometric score index was used to evaluate the degree of glomerulosclerosis. Tubulointerstitial fibrosis was assessed semiquantitatively.5. Immunohistochemical staining:Four micrometer-thick sections of 10% formalin fixed tissues were stained with antibodies as follows:mouse anti-α-SMA antibody and mouse anti-ED1 antibody. Briefly, sections were deparaffinized, washed with PBS, and incubated with 3%H2O2 in methanol to block endogenous peroxidase activity. Then, Sections were incubated overnight with the anti-a-SMA and anti-ED1 antibody in a humidified chamber at 4℃. The localization of the first antibody was visualized by an indirect immunoperoxidase method. All of these sections were examined in a masked manner using a light microscopy.6. Assessment of MDA levels in renal cortex:MDA levels were detected by the thiobarbituric acid method. Absorbance was measured at 532 nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the coomassie blue method.7. Assessment of superoxide dismutase activity:SOD activity was measured by the xanthine oxidase method. Absorbance was determined at 550 nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the coomassie blue method.8. Western blot analysis:Tissue samples from the kidneys were prepared with lysis buffer. Protein samples (30μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of protein was demonstrated by the ratio of integral optical density (IOD) between specific protein andβ-actin. 9. Real-time quantitative PCR:Total RNA from Tissue samples was extracted with Trizol, according to the manufacturer's protocol. The mRNAs were reverse-transcripted into cDNAs using cDNA synthesis kit. Real-time PCR was performed using Applied Biosystems TaqMan 7900HT detection system. The mRNA levels were estimated from the value of the threshold cycle (Ct) of the real-time PCR adjusted by that ofβ-actin through the formula 2ΔCt(ΔCt=gene of interest Ct-β-actin Ct)-(gene of control-β-actin Ct)Results1. Fenofibrate treatment attenuates the elevation in levels of urine albumin excretion rate without an apparent effect on blood pressure in SHRs:SHRs had an elevation in levels of urine albumin excretion rate compared with WKY rats and fenofibrate treatment significantly prevented the rise in these values. However, no differences in levels of serum creatinine and blood urea nitrogen were observed among these three groups. SHRs with or without fenofibrate treatment had significantly higher blood pressure compared with WKY rats. However, no significant differences in blood pressure were observed between SHRs with and without fenofibrate treatment. SHRs with or without fenofibrate treatment had significantly lower body weights compared with WKY rats.2. Fenofibrate ameliorates glomerulosclerosis and tubulointerstitial fibrosis in SHRs:PAS staining showed that segmental glomerulosclerosis was present only in a small number of glomeruli in the SHR group and absent in the WKY group. Moreover, Masson's trichrome staining showed significant tubulointerstitial fibrosis in SHRs. Fenofibrate administration significantly reduced the severity of glomerulosclerosis and tubulointerstitial fibrosis in SHRs. These observations were confirmed by quantitative analysis. Immunohistochemical staining for a-SMA showed that myofibroblasts in the glomeruli and tubulointerstitial space were significandly increased in SHRs and were markedly decreased by fenofibrate. 3. Fenofibrate prevents collagen expression and deposition in SHRs: Representative western blots showed that collagenⅣ, MMP-9 and TIMP-1 were significantly increased in the kidney of SHRs. However, fenofibrate treatment reduced the expression of collagenⅣ, MMP-9 and TIMP-1.4. Fenofibrate treatment reduces inflammatory cell accumulation in SHRs: Comapred with WKY group, MCP-1 mRNA levels were significantly increased in SHRs and was decreased by fenofibrate treatment. And, SHRs had a significant accumulation of ED1-positive cells in the glomeruli and tubulointerstitial space. Fenofibrate treatment markedly attenuated monocytes/macrophages accumulation in the kidney.5. Fenofibrate attenuates oxidative stress and increases renal SOD activity and mRNA levels in SHRs:Oxidative stress was evaluated by determination of the levels of MDA in the kidney, an index of lipid peroxidation. MDA levels were markedly increased in the kidney of SHRs and were decreased by fenofibrate treatment. Cu/Zn SOD activity and mRNA levels were significantly decreased in SHRs and was increased by fenofibrate treatment. However, Mn-SOD activity and mRNA levels were similar in these three groups. Protein levels of p47phox, a NAD(P)H oxidase subunits, in the kidney paralleled with MDA levels.6. Fenofibrate reduces TGF-β1 expression and p38 MAPK and JNK phosphorylation in SHRs:The effect of fenofibrate on TGF-β1 Expression and the phosphorylation of p38 MAPK and JNK were investigated. Representative western blots showed that TGF-β1 Expression, p38 MAPK and JNK phosphorylation were significantly increased in the kidney of SHRs and reduced by fenofibrate treatment. However, no differences in protein levels of total p38 MAPK and JNK were observed among these three groups.Conclusions1. Early renal injury in SHRs was characterized by mild proteinuria, glomerular sclerosis, tubulointerstitial fibrosis, inflammatory cells recruitment and normal renal function.2. PPARa agonist fenofibrate exerted renoprotective effects against hypertensive renal injury in SHRs.3. The renoprotective effects of PPARa agonist fenofibrate may be due to the inhibition of TGF-β1 expression and inflammatory cell recruitment through suppression of NADPH oxidase activity, upregulation of Cu/Zn SOD activity, and, thus, inhibition of phosphorylation of p38 MAPK and JNK.4. Fenofibrate, as a potent antioxidant, may have therapeutic potential for the treatment of oxidative kidney damage, such as hypertensive renal injury. BackgroudAortic aneurysms are permanent and localized aortic dilations defined as having diameters 1.5-times greater than normal. Important histological features of aneurysms include chronic adventitial and medial inflammatory cell infiltration, elastin fragmentation and degeneration, and medial attenuation. In the United States alone abdominal aortic aneurysms(AAA) affect 3% of individuals 60 years or older, necessitate 46,000 surgical interventions, and cause～15,000 deaths annually.1 Despite considerable descriptive knowledge of the pathomorphology of AAA, insufficient understanding of the molecular mechanisms underlying its pathogenesis currently limits the prevention and treatment of this human disease.Recent human studies from several groups indicate that human AAAs comprise an inflammatory disease characterized by the predominance of T helper cell type 2 (Th2) cytokine expression, especially IL-4 and the paucity of Th1 cytokines, especially interferon-γ(IFN-γ). Animal studies show that aortic allografts deficient in interferon-γ(IFN-γ) signaling developed AAA correlating with skewed Th2 cytokine environments, suggesting important regulatory roles for Th1/Th2 cytokine balance in modulating matrix remodeling and important implications for the pathophysiology of aortic aneurysm and atherosclerosis. Leptin, a cytokine-like hormone produced primarily by adipocytes, has been clearly demonstrated to play an important role in body weight regulation through effects on feeding and energy expenditure. In addition to central effects of leptin on appetite and metabolism, several studies have described direct leptin effects on immune cells, including the promotion of T lymphocyte type 1 helper (Th1) response, of potential importance to the process of atherosclerosis. Importantly, ob/ob mice have reduced secretion of IL-2, LFN-γ, TNF and IL-18, and increased production of TH2-type cytokines, such as IL-4 and IL-10, after mitogenic stimulation.Thus, we hypothesized that leptin, with the effect of regulation of Thl/Th2 cytokines, may have protective effect on the formation of AAA induced by AngⅡin ApoE-/- mice.Methods1. Animal models and experimental design:8 weeks old ApoE-/- mice were infused with saline or AngⅡ(1000 ng/kg/min) for 4 weeks and/or intraperitoneal injections daily with 0.6μg/g BW of recombinant murine leptin or vehicle control (n=8 per group).The dose of leptin chosen was based on a protocol used to achieve weight loss and fertility in leptin-deficient mice.2. Assessment of systolic blood pressure:Systolic blood pressure (SBP) was assessed every weeks throughout the study using a tail-cuff method in conscious rats after prewarming at 38℃for 10 min.3. Assessment of blood lipid:At the end of the study, the animals were anesthetized with pentobarbital, blood samples were collected from the right ventricle and serum was stored at -80℃for subsequently analyzing TG, TC, LDL-C and FFA.4,Measurement of abdominal aortic diameters and AAA incidence:Aortic diameters and AAA incidence were measured. The maximum width of abdominal aortas was measured with computerized morphometry. Aneurysm incidence was quantified on the basis of a definition of an external suprarenal aorta width that was increased by 50% or more compared with saline-infused mice. 5. Histopathological study:Coronal sections of renal tissue (3 to 4-μm-thick) were stained with Verhoeff-Van Giessen staining for analysis of elastin band rupture in AAA, and examined by light microscopy in a blinded fashion.6. Immunohistochemical staining:Four micrometer-thick sections of 10% formalin fixed tissues were stained with antibodies as follows:mouse anti-IL-4 antibody and mouse anti-INF-γantibody. Briefly, sections were deparaffinized, washed with PBS, and incubated with 3% H2O2 in methanol to block endogenous peroxidase activity. Then, Sections were incubated overnight with the anti- IL-4 and anti- INF-γantibody in a humidified chamber at 4℃. The localization of the first antibody was visualized by an indirect immunoperoxidase method. All of these sections were examined in a masked manner using a light microscopy.7. Western blot analysis:Tissue samples from the kidneys were prepared with lysis buffer. Protein samples (30μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of protein was demonstrated by the ratio of integral optical density (IOD) between specific protein andβ-actin.Results1. Leptin did not alter the development of hypertension in response to AngⅡ: ApoE-/- mice infused with AngⅡdeveloped moderate increases (25 mmHg) in systolic blood pressure during the 4 weeks of the study. This elevation in blood pressure was apparent within 3 days after pump implantation and was maintained throughout the course of AngⅡinfusion. Leptin did not affect systolic blood pressure in AngⅡ-infused mice.2. Leptin did not alter the concentrations of serum lipids:ApoE-/- mice infused with AngⅡhad an elevation in levels of serum TC and LDL-C. Leptin had no significantly effect on the rise in these values. No differences in levels of serum TG and FFA were observed among these three groups.3. Leptin markedly attenuated AngⅡ-induced hypertensive AAAs:The incidence of AAAs in the suprarenal aorta of Angll infused mice was 87.5%. intraperitoneal injections daily with leptin decreased the incidence (85% vs 62.5%, AngⅡalone vs AngⅡ+ leptin, respectively; P<0.01; Table 2) and the severity of aneurysms formed by AngⅡ(Figure 4).4. Leptin markedly decreased the protein levels of MMP-2 and MMP-9 in AAA:Because matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a critical role in AAA formation, we examined MMP protein expression in abdominal aortic tissue homogenates. AngⅡinfusion induced an increase in the protein expression of both MMP-2 and MMP-9 compared with control. Treatment with leptin markedly decreased the protein levels of MMP-2 and MMP-9 in abdominal aortae of AngⅡ-infused mice.5. Leptin markedly decreased Th2 cytokine IL-4 expression and increased Th1 cytokine INF-γexpression in AAA:Representative western blots showed that IL-4 expression was significantly increased in AAA induced by AngⅡand reduced by leptin treatment. However, INF-γexpression was significantly increased in AAA by by leptin treatment. The result from immunohistochemical staining paralleled with that from western blot.Conclusions1. Infusion with Angll induced the formation of hypertensive AAAs in ApoE-/-mice.2. Leptin markedly attenuated AngⅡ-induced hypertensive AAAs in ApoE-/-mice with no effect on serum lipids and systolic blood pressure.3. The protective effects of leptin on hypertensive AAAs may be due to the inhibition of Th2 cytokine IL-4 expression and induction of Th1 cytokine INF-γ expression and thus inhibition of the expression of MMP-2 and MMP-9.4. Leptin, with the effect of regulation of Th1/Th2 cytokines, may have therapeutic potential for the treatment of hypertensive AAA.