| Part OneThe mechanism of cofilin-1 on hypertension-induced renal damageBackgroundKidney is one of the mainly involved targeting organs of hypertensive damage. About 18% of patients with hypertension have eventually developed kidney failure, while 4%-16% of patients with antihypertensive treatment have developed sustained proteinuria in recent years. Pathogenesis of hypertensive renal disease is very complex, including activation of renin-angiotensin-aldosterone system, increases of salt uptake, imbalance of vasoactive substance and genetic factors. All above factors participate in hypertensive renal damage. Inflammation and these factors are closely linked, cause- and -effect. It participates in the development of hypertensive nephropathy. As an ubiquitous nuclear transcription factor, NF-κB activates in the kidney diseases and then leads to secretion of inflammatory mediators. Inactive NF-κB is sequestered within the cytoplasm. The various stimuli activate NF-κB and cause the migration of NF-κB from cytoplasm to the nucleus, finally activate the transcriptions of several target genes, including monocyte chemotactic protein 1(MCP1) and Interleukin-1β (IL1β). The translocation of RelA/p65 (an NF-κB subunit) from cytoplasm to the nucleus requires dynamic alterations. The study of actin filaments system is a new perspective on the study of hypertensive renal damage.Cofilinl is an important actin-binding protein that plays an essential role in regulating actin filament dynamics and reorganization by stimulating the severance and depolymerization of actin filaments. It participates in a variety of physiological processes. It has been reported that cofilinl is involved in nuclear translocation of NF-κB in endothelial cells. However, its roles in renal tubular epithelial cells and hypertensive nephropathy model are remains unknown. Therefore, we will investigate the underlying mechanisms of cofilin-1 in the development of hypertensive nephropathy via molecular biology technologies.Objectives1. To observe the characteristics of renal histological changes in SHR, to research the expressions of cofilinl, inflammatory mediators and NF-κB.2. To stuy the inflammation and cofilinl activity in AngⅡ-induced HK-2 cells.3. To explore the molecular mechanisms of cofilinl on hypertensive renal damage.MethodsAnimal experiment21-week-old male SHRs (n=20) and Wistar-Kyoto rats (WKYs) (n=10) were randomly divided into three groups (n=10 per group):SHR-C (a SHR control group administered 1 mL 0.9% sodium chloride orally), WKY-C (a WKY control group administered 1 mL 0.9% sodium chloride orally), SHR-H (SHRs administered GSPE at a dose of 250 mg kg-1 day-1). We selected grape seed proanthocyanidins extract (GSPE) as a drug control. The animals were subjected to drug treatments as indicated above via oral gavage for 22 weeks. Tail systolic blood pressures (SBP) were measured weekly until the end of the experiment (22 times in total) in conscious rats using a Softron tail-cuff BP-2006A non-invasive sphygmomanometer. The rats were housed in individual metabolic cages for the collection of urine at 2-week intervals. The urine was centrifuged and stored at -80℃. Treatment was continued for 22 weeks, at which time the rats were weighed and anaesthetized using chloral hydrate.Cell clutureHuman renal proximal tubular cells (HK-2) were maintained in DMEM/F12 medium supplemented with 10% foetal bovine serum. HK2 cells were preincubated in either the presence or the absence of GSPE (50μg.mL-1) for 12 h before being stimulated either with or without AngⅡ (10-6mol.L-1) for 12 h. Then expressions of MCP1, IL-1β and nuclear translocation of NF-κB were observed in order to explore the pro-inflammatory effect of angiotensinⅡ and therapeutic effect of GSPE. Lentiviral-shRNA specific for interfering cofilin-1 expression, recombinant lentiviral Lv-cfl-shRNA and a nonspecific lentiviral control Lv-con-shRNA were obtained from GeneChem. The cells were transfected with lentiviral suspension using transfection reagent according to the manufacturer’s recommendations. Following 72-96h, transfection efficiency was measured by testing the expression ratio of eGFP via fluorescence microscopy. Moreover, the knockdown effect of cofilinl was evaluated via Western blotting. After transfection and passage, cells were challenged for 12 h with AngⅡ (10-6mol.L-1). Effects of cofilin-1 knockdown on Angiotensin Ⅱ-induced formation of fiber stress, RelA/p65 nuclear translocation, NF-κB activity and inflammatory mediators were investigated.1. Blood was collected from the abdominal aorta, serum creatinine and blood urea nitrogen were then detected.2. Changes of renal morphology and histology were measured by HE staining.3. The expressions of IL-1β, MCP1 and NF-κB were measured by immunohistochemical analysis;4. Renal protein levels of IL-1β and MCP1 were measured via commercial ELISA kits.5. Immunofluorescence staining was used to observe the distribution of NF-κB p65.6. The cells were incubated with rhodamine-phalloidin to localize the F-actin filaments.7. NF-κB luciferase reporter assay was used to detect NF-κB activity.8. Western blot was used for measurements of cofilinl, p-cofilinl, NF-κB p65, MCP1, IL-1β in renal tissues and HK-2 cells.Results1. Body weight and systolic blood pressure(SBP) of ratsAt the end of the experiment, body weight did not differ among the four groups. Compared with the age-matched WKYs, SBP of SHRs was significantly increased(P<0.05). SBP of SHRs increased with age, no significant difference between SHR-C and SHR-H (p>0.05). The result indicated that GSPE treatment failed to attenuate the high blood pressure of SHRs in the present study.2. Serum creatinine, blood urea nitrogen of ratsThere were not significantly differences of serum creatinine and urea nitrogen among the groups (P> 0.05).3.24h-urine protein of ratsCompared with WKY-C group, the total urine protein of SHR was significantly increased (P<0.01); Compared with SHR-C group,24-h urine protein level of SHR-H was significantly decreased (P<0.01)4. Renal pathological changesCompared with WKYs, more extensive tubulointerstitial inflammatory cells infiltration and tubular edema in SHRs were observed. A quantitative analysis of HE staining demonstrated significant interstitial inflammatory infiltration (including neutrophils, lymphocytes and monocytes) in the kidneys of SHR-C group compared with the SHR-H and WKY-C rats(P<0.05). GSPE administration significantly alleviated the inflammatory infiltration in the renal interstitial.5. Renal inflammatory mediators and NF-κB activityCompared with WKY-C, the renal protein levels of MCP1 and IL-1β were significantly increased in SHRs(P<0.01). Compared with SHR-C, they were significantly decreased in SHR-H(P<0.01), which indiates that GSPE treatment can improve renal inflammation. Immunohistochemical staining of MCPland IL-1(3 showed similar results. Immunohistology staining for NF-κB p65 demonstrated that a small quantity of NF-κB p65 localized in the cytoplasm of the tubular epithelial cells in the WKY rats and that the nuclei were relatively unstained. By contrast, NF-κB p65 expression was increased in the cytoplasm and nuclei of the SHR kidneys. NF-κB p65 localized primarily in the nuclei of the renal tubular epithelial cells, a finding indicative of its increased activity, particularly in the SHR-C group. Treatment with high dose GSPE decreased NF-κB activity in SHR kidneys (P<0.01,).6. Renal cofilin1 activity and Iκ-Bα expressionThere were no significant differences of total cofilin1 level among the four groups. But the protein content of p-cofilin1 was significantly increased in the SHRs, which indicated that the activity of cofilinl was significantly decreased. Additionally, the activity of cofilinl was significantly increased by GSPE treatment(P<0.05). Compared with WKY-C rats, Iκ-Bαin SHR groups were decreased(P<0.05). Compared with SHR-C, SHR-H was somewhat increased (P<0.05).7. The protein expressions of MCP-1, IL-1β, cofilinl and p-cofilinl in AngⅡ-induced HK2 cellsWestern blotting was performed to examine the protein expressions of MCP1 and IL-1β in vitro. AngⅡ treatment for 12 h significantly increased the protein levels of MCP-1 and IL-1β compared with the control group in the HK2 cells (P<0.01). Additionally, stimulation with AngⅡ promoted p-cofilinl expression, resulting in cofilinl inactivation. GSPE (50μg.mL-1) significantly reduced the levels of the above-mentioned proteins in the AngⅡ-treated HK2 cells (P<0.05).8. The effects of cofilinl knockdown on angiotensinⅡ-induced stress fibre formation in HK2AngⅡ stimulation led to increased formation of stress fiber in HK-2 cells transfected with the Lv-cfl-shRNA and blank virus Lv-con-shRNA, efficiency of transfection and interference were both higher than 90%. Transfection with Lv-cfl-shRNA caused excessive formation of stress fiber. Western blot showed that both cofilinl interference and AngⅡ stimulation can induce higher expression of F-actin. While the highest expression of F-actin is in Lv-cfl-shRNA+AngⅡ group.9. The effects of cofilinl knockdown on angiotensinll-induced NF-κB p65 nuclear translocation in HK2An analysis of nuclear extracts via immunoblotting demonstrated that AngⅡ-induced NF-κB p65 translocation into the nucleus was attenuated by either GSPE or cofilinl depletion, which increased the level of NF-κB p65 in the cytoplasm. AngiotensinⅡ stimulation resulted in reduction of IκB-α protein level in the cytoplasm; however, said reduction was significantly reversed by cofilinl depletion or GSPE. Luciferase reporter gene assay was used to measure NF-κB activity in different groups. Compared with the control group, NF-κB activity of other groups was significantly increased (P<0.01). The NF-κB activity was lowest in Lv-cfl-shRNA+ AngⅡ group.10. The effects of cofilinl knockdown on protein expressions of MCP-1, IL-1β in AngiotensinⅡ-induced HK2 cellsAngⅡ stimulation for 12 h significantly increased the levels of MCP-1 and IL-1β compared with the control group, as demonstrated via the Western blotting analysis. Cofilinl deletion significantly reduced said protein levels in the Angll-treated HK2 cells. The inhibitory effects on inflammatory mediators were equal between cofilinl knockdown and GSPE pretreatment.Conclusions1. The increase of NF-κB activity and decrease of cofilinl activity simultaneously occurred in kideys of SHRs.2. AngiotensinⅡ induced high expression of inflammatory mediators and inactivation of cofilinl.3. Cofilinl is involved in hypertensive nephropathy by modulating the nuclear translocation of NF-κB and the expression of its downstream inflammatory factors in renal tubular epithelial cells.Part TwoThe renoprotective effect of grape seed proanthocyanidin extract on hypertension-induced renal damageBackgroundOxidative stress is the earliest pathological changes in kidney of hypertensive models, which is present throughout the entire course of hypertension. NADPH oxidases on mesangial cells and renal tubular epithelial cells were activated by increased angiotensinⅡ and then a great many of reactive oxygen species(ROS) were produced. Activation of intra-renal redox-sensitive signaling pathways is associated with glomerular damage, proteinuria, excessive sodium and water retention as well as nephron loss. In addition to these mechanisms, oxidative stress also induces inflammation and fibrosis in the kidney, which ultimately facilitates the development of hypertension. Given the key role of oxidative stress, more and more studies are conducted on antioxidants therapy in hypertensive renal damage. Proanthocyanidins are naturally occurring compounds found widely in plants. The seeds of the grape are particularly rich of proanthocyanidins. Grape seed proanthocyanidins have been shown to be potent antioxidants and free radical scavengers, being more effective than either ascorbic acid or vitaminE. Studies have showed that grape polyphenols can reduce blood pressure and cardiac hypertrophy, while increase vascular compliance in SHRs. GSPE has also been found to improve renal injury in diabetic rats.In view of the antioxidant and anti-inflammatory effects of GSPE, it was used as an anti-inflammatory drug in SHRs and angiotensinⅡ-induced HK-2s in part one. Its protective effect in kidney was remarkable. In order to fully assess the protective effects of GSPE in hypertensive renal damage, we carried out this part research.Objectives1. To observe the effect of GSPE on histological lesions in SHRs.2. To study the effect of GSPE on renal inflammation and fibrosis in SHRs.3. To investigate the effect of GSPE on renal oxidative stress in SHRs.MethodsAs the same of animal experiments in part one, but only a SHR-L group was added. In this group, GSPE were given at a dose of 100 mg kg-1 day-1 in SHRs. The animals were subjected to drug treatments as indicated above via oral gavage for 22 weeks. Tail systolic blood pressures (SBP) were measured weekly until the end of the experiment (22 times in total) in conscious rats using a Softron tail-cuff BP-2006A non-invasive sphygmomanometer. The rats were housed in individual metabolic cages for the collection of urine at 2-week intervals. The urine was centrifuged and stored at-80℃. Treatment was continued for 22 weeks, at which time the rats were weighed and anaesthetized using chloral hydrate.1. Systolic blood pressure (SBP) and body weight were measured in conscious animals before the start of treatment and weekly during treatment.2. Blood was collected from the abdominal aorta, serum creatinine and blood urea nitrogen were then detected. Urinary supernatant was used to detect urinary creatinine, 24h urinary protein and microalbumia.3. Changes of renal morphology and histology were measured by the staining of HE, PAS and Masson. Then ultra-thin sections stained with uranylacetate and lead citrate were examined under an H-800 transmission electron microscope.4. The protein levels of IL-6, TNF-a in renal tissues were measured by ELISA kits, the mRNA levels of IL-6, TNF-a were measured by RT-PCR.5. Immunohistochemistry was used to observe the expressions of TGF-β1/α-SMA/collagenⅣ in renal tissues.6. Western blot was used to explore the expression of p-p65.7. H2O2, MDA contents and the activities of antioxidant enzyme CAT, SOD in renal homogenates were measured by commercial kits.Results1. Effect of GSPE on body weightBody weight of all rats gradually increased as time passed. But there was no difference among the four groups (P>0.05).2. Effect of GSPE on systolic blood pressureCompared with the WKY, the mean arterial blood pressure of the SHR showed a significant increase thoughout the study (P<0.05). Blood pressure of SHR gradually increased with time, but 22-week treatment with GSPE could not lower arterial blood pressure of SHR.3. Effect of GSPE on renal functionCompared with WKY-C group, urinary protein and microalbumin of 24h in SHRs were both significantly increased(P<0.01). Compared with SHR-C group, urinary protein of 24h in SHR-L was significantly decreased(P<0.01). But there was no difference between SHR-H and SHR-L(P>0.05). As for 24h urinary microalbumin, treatment with high dose GSPE decreased its level in SHR kidneys (P< 0.01),while low dose has no significantly effect. There were no statistically difference of creatinine clearance among the four groups (P> 0.05).4. Effect of GSPE on morphological changes in renal tissueThe WKY-C group showed edema in individual renal tubular epithelial cells via HE staining. Edema of renal tubular epithelial cells and infiltration of inflammatory cells in renal tubuar interstitial were observed in kidneys of SHR-C. Treatment with GSPE significantly alleviated edema and infiltration of inflammatory cells.As demonstrated by Masson’s trichrome staining, large areas of fibrosis were detected in kidney of SHR-C and SHR-L, which displayed a significantly geater renal collagen deposition than that observed in the WKY-C group. The fibrotic changes in the kidney were significantly reduced in SHR-H received a 22-wk treatment with GSPE.The representative renal histology of periodic-acid Schiff (PAS)-stained sections was shown. Compared with age-matched WKY rats, the glomerular accumulation of PAS-positive matrix was evident in SHR-C rats. Matrix expansion was significantly lower in the GSPE-treated group than in the untreated group.Glomerular ultrastructure was examined by electron microscopy. Compared with WKY rats, we observed focal fusion of foot processes, thickening of the glomerular basement membrane, increased cytoplasmic vesicles of podocytes in control SHR rats. SHR-L group showed focal thickening of the basement membrane and deformation of foot processes. SHR-H group showed almost normal glomerular filtration barrier.5. Effect of GSPE on inflammatory markersProtein levels of IL-6 and TNF-a were higher in kidneys of SHR. Following GSPE* treatment, the upregulated IL-6 and TNF-a protein levels were decreased in the SHR-H and SHR-L groups. The reduction effect was more significant in high dose group SHR-H(P< 0.05). Then we further verified the changes by qPCR. Next, we measured the levels of phosphorylated p65 and total p65 by westen blot. Western blot showed that the protein levels of p-p65 were significantly increased in SHRs compared with WKY-C(P<0.01). Protein levels of p-p65 gradually reduced from SHR-C, SHR-L to SHR-H.6. Effect of GSPE on fibrosis markersTo evaluate the effect of GSPE treatment on the fibrosis in the model we examined expressions of collagenⅣ, a-SMA and TGF-β1 by immunohistochemistry. Immunohistochemical staining showed that TGF-β1, collagenⅣ and a-SMA in SHRs were higher than WKYs. After GSPE treatment, TGF-β1 and collagen Ⅳwere significantly reduced (P<0.01), but the reduction of TGF-β1 was not significant between SHR-L group and SHR-H group (P>0.05). As for a-SMA, only high doses GSPE treatment group showed decrease (P<0.05).7. Effect of GSPE on oxidative stressSHRs had lower SOD and CAT activities than WKYs (P<0.05). After long time treatment with GSPE, the downregulated SOD, CAT levels were increased in SHRs, especially refered to SOD (P<0.05). By contrast, H2O2 and MDA expressions of SHRs were higher than in kidneys of WKYs (P<0.05). After 22-week treatment by GSPE, the level of MDA was markedly decreased. The decrease was in a dose-dependent manner. |
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