摘要
慢性肾脏病(CKD)是一种常见肾脏疾病,若未能进行有效治疗,最终将进入终末期肾脏病(ESRD)。肾纤维化是各种病因导致的CKD发展至ESRD阶段的共同途径和主要病理结果。氧化应激、慢性炎症及转化生长因子β(TGF-β)等细胞因子均在其发生发展中发挥重要作用,但其确切发病机制尚不清楚。Kruppel样因子15(KLF15)是一种在体内广泛分布的核转录因子,属于锌指蛋白家族,通过对多种基因的表达调控参与细胞分化、增殖、凋亡等生物学过程。2007年Fisch S等研究表明,KLF15参与调控心脏纤维化的发生,其机制与抑制I型胶原转录和结缔组织生长因子(CTGF)启动子活性有关。肾纤维化的发生与心脏纤维化存在相似机制,但目前尚无KLF15与肾脏纤维化关系的研究,其是否是影响CKD肾纤维化发生及进展的重要因素是本研究的关注方向。
限制蛋白饮食是CKD的主要治疗策略之一,可减轻CKD患者氮质血症,延缓肾衰病情进展,但单纯低蛋白血症可能会引起营养不良等问题。目前临床常采用低蛋白加α酮酸治疗CKD患者,α酮酸可提供必需氨基酸并减少氨基氮的摄入,在不增加体内毒素的同时维持一个相对良好的营养状态。但目前仍不清楚低蛋白加α酮酸饮食在延缓CKD病程进展中的作用是否要优于单纯低蛋白饮食及其具体机制。考虑到氧化应激、炎症在CKD肾纤维化中的作用,本研究的目的在于：明确α酮酸加低蛋白饮食是否较单纯低蛋白饮食可更好地延缓CKD大鼠肾损害进展,减轻氧化应激、和炎症在其中的作用,此外,我们还将进一步探讨KLF15在肾纤维化进展中的作用和机制。
为此,我们首先建立5／6肾切除SD大鼠CKD模型,对照组大鼠行假手术(Control,n=12),手术组大鼠分别给予正常蛋白饮食(NPD, n=15)、低蛋白饮食(LPD, n=15)和低蛋白饮食加α-酮酸(LPD+KA, n=15),干预6个月留取血、尿标本后处死大鼠,进行血、尿生化检测；留取肾组织包埋切片,进行Masson染色、电镜、I型胶原和纤维连接蛋白免疫组织化学检测；提取肾组织总RNA,进行Real-time PCR检测TGF-β1、Ⅰ型胶原、Ⅲ型胶原、Ⅳ型胶原和纤维连接蛋白的表达。结果显示,血尿素氮、尿蛋白、Masson染色病理评分、电镜、胶原染色和纤维化相关的Real-time PCR均显示：与对照组相比,NPD组出现明显的肾功能恶化,LPD较NPD可改善病情进展,而LPD+KA对肾功能的保护作用较LPD更明显(P<0.05)。LPD组出现低体重、低血清白蛋白等营养不良的表现,而LPD+KA组未出现明显的营养不良(P<0.05)。由于已有研究证实,蛋白营养不良可致组织氧化应激和炎症水平的增加,而氧化应激和炎症是CKD肾纤维化的发生和进展的重要因素,因此,低蛋白加α酮酸饮食较单纯低蛋白饮食更好地减缓肾损害进展,其作用可能与改善营养不良,减少氧化应激和炎症反应有关。
为进一步证实氧化应激和炎症在低蛋白加α酮酸饮食在延缓CKD病程进展中的作用,我们进一步检测了各组的氧化应激和炎症的相关指标。将上述大鼠肾组织行组织匀浆检测SOD、MDA,提取总蛋白行oxybolt检测蛋白氧化水平,同时组织切片进行硝基化酪氨酸和CD68免疫组织化学检测。此外还提取肾组织总RNA,采用Real-time PCR检测TNF-α, MCP-1、RANTES和CXCL-1的mRNA表达。结果显示,LPD组和LPD+KA组氧化应激(MDA、硝基化酪氨酸、oxybolt)和炎症(CD68、TNF-α、MCP-1、RANTES、CXCL-1)相关指标较NPD组显著改善,同时,LPD+KA组相关指标也显著好于LPD组(P<0.05)。这些结果表明,低蛋白加α酮酸饮食通过减轻氧化应激和炎症反应而减缓CKD肾损害进展。
随后,我们研究了KLF15在肾纤维化中的作用。首先通过对上述大鼠肾组织的免疫荧光和Real-time PCR检测发现,KLF15主要在对照组肾组织的系膜细胞和间质成纤维细胞中表达,5／6肾切除后表达显著下降,予以低蛋白和低蛋白加α酮酸饮食可使其表达增加,且LPD+KA组较LPD组增加更为显著(P<0.05)。体外实验中我们采用TGF-β1 TNF-α和H202干预大鼠系膜细胞(RMCs),提取总RNA行Real-timePCR检测发现干预后KLF15表达降低(P<0.05)；同时通过对TNFR1-/-小鼠系膜细胞及NF-κB基因敲除的RelA-/-小鼠胚肾间质成纤维细胞(MEF)进行TNF-α干预,Real-time PCR检测发现TNF-a通过TNF受体1和NF-κB途径减少KLF15表达。为进一步研究KLF15与纤维化的关系,我们将KLF15质粒瞬时转染人胚肾细胞(HEK293)和RMCs细胞,提取总RNA行Real-time PCR和提取细胞蛋白及上清行ELISA检测,结果显示过表达KLF15可显著减少HEK293和RMCs细胞纤维连接蛋白和IV型胶原的表达(P<0.05)。此部分结果表明KLF15参与调控肾纤维化进程,饮食干预可通过影响KLF15表达而延缓CKD进展。
总之,本研究发现α酮酸加低蛋白饮食较单纯低蛋白饮食可显著地延缓CKD大鼠肾损害进展,其作用与降低残余肾组织的氧化应激和炎症反应有关。KLF15表达受氧化应激、炎症等多种因素影响,并参与调控CKD肾纤维化进程,饮食干预可通过影响KLF15表达而延缓肾损害进展。
Chronic kidney disease (CKD) is one of the most common kidney diseases and may cause end stage renal disease without effective therapeutic interventions. Renal fibrosis is the result of extracellular matrix accumulation and a hallmark of progressive CKD. Multiple factors, including oxidative stress, chronic inflammation and transforming growth factorβ(TGF-β), are all implicated in the development and progression of renal fibrosis, but the exact mechanisms of CKD remain unknown. Kruppel-like factor 15 (KLF15) is a zinc finger-containing transcription factor and plays an important role in regulation of differentiation, proliferation and apoptosis. In 2007, Fisch S et al reported that KLF15 may play an important role in heart fibrosis, and further study indicated that the levels of type I collagen transcription and the activity of connective tissue growth factor (CTGF) promoter were decreased by KLF15. As heart fibroses share similar mechanisms, KLF15 may also be an important factor in renal fibrosis.
Dietary protein restriction is a major therapy in CKD. Lowering dietary protein intake to slow down the progression of renal failure would decrease the metabolic burden, especially the levels of urea nitrogen. Since malnutrition is a concern for long-term protein restriction, ketoacida, a nitrogen free substitution for the essential amino acids, have been prescribed together with low protein diet to CKD patients. Ketoacids are equally efficient as their essential amino acids counterpart in protein synthesis. Furthermore, they reduce accumulation of nitrogen waste products by decreasing exogenous nitrogen supply and enhancing reutilization of stored nitrogen. However, it is still unclear whether low protein diet supplemented with ketoacids is better than low protein diet alone in renal protection of CKD patients, and the mechanisms by which they decrease the progression of CKD are unknown. Knowing that oxidative stress and inflammation are implicated in the development of renal fibrosis, we hypothesized that low protein diet supplemented with ketoacids might offer a better protective effect than low protein diet alone. We further evaluated the effect of diet treatment on the level of KLF15 expression and explored the mechanisms in the progression of renal fibrosis in a CKD rat model.
Forty-five 5/6 nephrectomy SD rats were equally randomized into three groups:NPD group (22% protein); LPD group (6% protein), and LPD+KA group (5% protein+1% ketoacids), all for 24 weeks. Sham operated rats (n=12) were used as control. Blood and urine samples were collected for biochemical test. Remnant or control kidneys were removed, embedded and sliced in sections, and stained with Masson's, type I collagen and fibronectin immunohistochemical staining as well as electron microscope study. Total RNA was extracted from renal tissue and expressions of fibrosis-related genes including TGF-β1, typeⅠ, type III, type IV collagen and fibronection were examined by real-time PCR. BUN, Proteinuria, decreased renal function, glomerular sclerosis and tubulointerstitial fibrosis were observed in the remnant kidneys of NPD group. Protein restriction ameliorated above changes, and the effect was more obvious in LPD+KA group (P<0.05) than LPD group. Lower body weight and serum albumin level were found in LPD group, indicating protein malnutrition (P<0.05). Since protein malnutrition has been reported to cause oxidative stress and inflammation, which are implicated in the development and progression of renal fibrosis, it seems that low protein diet supplemented with ketoacids plays a more protective role than low protein diet alone in protecting remnant kidney function against progressive injury. The effect may be mediated by ameliorating malnutrition, thus protecting the remnant kidney against oxidative stress and inflammation injury.
Levels of oxidative stress and inflammation were further measured to convince the role that they played in protecting CKD renal function by low protein diet supplemented with ketoacids. The results of MDA, oxybolt and nitrotyrosine immunohistochemical staining showed that renal lipid and protein oxidative products, and chronic inflammation as characterized by extensive macrophage infiltration and increased mRNA expression of cytokine and chemokines (TNF-a, MCP-1, CXCL-1 and RANTES) were substantially reduced in LPD and LPD+KA groups, and the reduction in LPD+KA group was more pronounced than that in LPD group (P<0.05). These findings indicate that low protein diet supplemented with ketoacids played a better role in blocking the progression of renal pathology of CKD by mediating oxidative stress and inflammation injury.
Then, we studied the role of KLF15 in renal fibrosis and further explored the underlying mechanism. Immunofluorescence revealed that large numbers of KLF15 positive cells were present in both glomeruli and interstitial of the control group. The levels of KLF15 mRNA and the number of KLF15 positive cells were drastically decreased in remnant kidney of 5/6 nephrectomized rats. While restriction of protein intake partially restored the levels of KLF15, the effect was more pronounced in LPD+KA group than that in the groups using dietary protein alone (P<0.05).It was found that in vitro mRNA levels of KLF15 in rat mesangial cells (RMCs) were decreased by TGF-β1, TNF-a, as well as H2O2 (P<0.05) Since the suppressive effect of TNF-a on KLF15 mRNA expression was largely lost in cells lacking TNF receptor 1 (mesangial cells from TNF receptor 1 knock-out mice TNFR1-/-) or p65 subunit of NFκB (embryonic fibroblasts from NFκB p65 deficient mice RelA-/-), both TNF receptor 1 and NFκB are likely required for TNF-a mediated inhibition of KLF15 expression. For further study of KLF15, RMCs and human embryo kidney cells (HEK293) were transfected with a KLF15 cDNA expression vector, and total RNA, cellular protein and culture supernatant were collected for Real-time PCR or ELISA study. It was found that overexpression of KLF15 was associated with a significant decrease in type IV collagen and fibronection expression (P<0.05), suggesting that KLF15 may also have a role in extracellular matrix regulation in renal fibrosis, and the improvement of renal disease by dietary treatment was associated with a partial restoration of KLF15 expression in remnant kidney.
In conclusion, the results of our study indicate that low protein diet supplemented with ketoacids plays a more renal protective role than low protein diet alone in the 5/6 nephrectomy rat model. The effect may be mediated by ketoacids through ameliorating oxidative stress and inflammation injury in remnant kidney tissue. The expression of KLF15 could be affected by oxidative stress, inflammation and multiple factors, and it may also play a role in extracellular matrix regulation in renal fibrosis in CKD. The improvement of renal disease by dietary treatment was associated with a partial restoration of KLF15 expression in remnant kidney.
引文
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