高亲和力钠依赖二羧酸转运蛋白（NaDC3）促进肾脏衰老的机制研究
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摘要
目的：肾脏功能随年龄增长逐渐下降，老年人终末期肾脏疾病(endstage renal disease，ESRD)的发生率高，且治疗有效率低，死亡率高。因此研究肾脏衰老的机制和延缓肾脏衰老的策略有重要意义。肾脏衰老的病理学特征是肾小球硬化、肾小管萎缩、肾间质纤维化和动脉内膜纤维性增厚，其中肾间质-小管的损伤对肾功能的影响更为重要。肾小管的重要功能之一就是主动吸收有用物质或分泌有害物质，而这些过程均需要消耗能量。近年来的研究表明能量代谢与衰老关系密切，而且限制热量摄入可延长很多物种的寿命，并可延缓衰老相关疾病的发生。与其他器官一样，肾小管的主要能量来源也依赖于三羧酸循环。有趣的是肾小管上皮细胞基底膜侧存在一种高亲和力钠依赖二羧酸转运蛋白(NaDC3)，其主要功能是将三羧酸循环的中间产物二羧酸与三羧酸从组织或血液中转运至肾小管上皮细胞内，为其提供能量合成的底物。我们实验室前期研究发现NaDC3在人和大鼠肾脏的表达随着衰老逐渐增加，在肾脏衰老中起重要作用。过表达NaDC3基因，可使人二倍体成纤维细胞(WI38细胞)提前出现衰老表型。但是NaDC3促进衰老的具体机制尚不清楚，我们推测与能量代谢有关。目前越来越多的证据表明限制能量摄入延缓衰老、延长寿命与Sir2有关。Sir2是三羧酸循环中的辅酶尼克酰胺腺嘌呤二核苷酸(NAD)依赖的组蛋白去乙酰化酶，它通过NAD把代谢与寿命联系在一起，最近研究发现SIRT1(Sir2在哺乳动物中最近的同系物)对肾脏有保护作用。那么NaDC3是否通过调控三羧酸循环中间产物而影响NAD的水平，进而影响SIRT1的表达和／或功能而促进衰老呢?为此本研究以能量代谢为切入点，首先观察SIRT1在不同月龄大鼠肾脏的表达情况及其与NaDC3表达的相关性；进一步研究了在人肾小管上皮细胞(HKC)和WI38细胞中NaDC3对NAD~+／NADH比值的影响及其与SIRT1表达和功能的关系，并且观察了NaDC3对SIRT1表达影响与WI38细胞衰老表型的关系。
方法：取1日龄、3月龄、12月龄和24月龄大鼠的肾脏组织，观察NaDC3和SIRT1的表达及SIRT1的活性变化，并分离培养各时间点大鼠肾小管上皮细胞，用免疫荧光法检测SIRT1的定位和表达变化。应用逆转录病毒介导的基因转移技术，将NaDC3基因分别转移至HKC和WI38细胞内，检测不同水平的NaDC3对SIRT1表达和活性的影响。在WI38细胞转染正义NaDC3后，检测衰老的相关指标，包括用β-半乳糖苷酶活性(SA-β-gal)染色方法检测了衰老相关SA-β-gal染色阳性率，用MTT法检测了细胞增殖能力，用流式细胞仪检测了细胞周期的变化，并计数了细胞分裂的代数等。在转染正义NaDC3的同时，给与SIRT1的激动剂白黎卢醇或限食大鼠血清，观察对上述衰老相关指标的影响，以证实NaDC3是否通过SIRT1影响了衰老的进程。同时检测了NaDC3对WI38细胞NAD~+／NADH比值的影响，并给与NAD刺激转染了正义NaDC3的WI38细胞，使NAD~+／NADH比值升高，观察衰老相关的指标变化，以证实NaDC3通过降低NAD~+／NADH比值，抑制SIRT1，继而加速衰老。
结果：随大鼠月龄增长，NaDC3在肾脏的表达水平逐渐增高，而SIRT1的表达和活性均逐渐下降。SIRT1在肾小管上皮细胞核内表达，荧光强度随大鼠增龄逐渐减弱。HKC和WI38细胞转染正义NaDC3后明显抑制了SIRT1的蛋白表达和去乙酰化酶活性；HKC转染反义NaDC3后SIRT1的蛋白表达和去乙酰化酶活性有所升高。在WI38细胞转染正义NaDC3后，加速了细胞衰老，表现为细胞增殖能力下降，SA-β-gal染色阳性率明显增高，细胞分裂代数减少，停滞在G1的细胞数量增多；给与SIRT1的激动剂白黎卢醇或限食大鼠血清，则能抑制细胞出现以上衰老相关的表型。转染正义NaDC3的WI38细胞NAD~+／NADH比值降低，给与NAD刺激，提高NAD~+／NADH的比值后，细胞衰老相关的表型部分得到抑制。
结论：在大鼠肾脏中，SIRT1的表达和活性随着增龄而下降，在肾脏衰老中起重要作用。NaDC3可通过增强能量代谢而抑制NAD~+依赖的SIRT1的蛋白表达和酶活性而促进肾脏衰老。
Background and objective: Normal aging alters renal function, the high frequency of end-stage renal disease (ESRD) in the elderly. The incidence of acute renal failure is higher in older subjects, and the clinical outcome of renal failure is significantly worse with regard to mortality, requirement of dialysis, and complete recovery. So it is important to study the mechanism of aging kidney. The pathological changes of tubular-interstitial are especially important because they determine the aggravate degree of renal function. High-affinity sodium-dependent dicarboxylate cotransporter (NaDC3) is expressed in basolateral membrane of proximal renal tubular epithelial cells primarily involved in the uptake and metabolism of Krebs cycle intermediates, which include a variety of di- and tricarboxylates such as succinate, citrate, and a-ketoglutarate. It induces the aging process. The pathway of NaDC3 mediating aging would provide the targets of delaying aging. But the mechanism is yet unclear. NaDC3 provides energy for renal tubular epithelial cells, and there are tight link between energy metabolism and lifespan. SIRT1, a NAD-dependent histone deacetylase, extends many organism lifespan and reduces aging-related diseases in caloric restriction. SIRT1 links energy metabolism and lifespan through NAD. We presumed NaDC3 probably regulated lifespan through inhibiting SIRT1 function. To determine the mechanism of aging induced by NaDC3, the study designed to investigate firstly the change of SIRT1 expression and activity in kidney with aging, and then detected the effects of NaDC3 on the level of SIRT1 expression and activity in HKC and WI38 cells, senescent-related phenotypes in WI38 cells, and NAD~+/NADH ratio.
Methods: The kidneys from different month-old rats were used to investigate the change of SIRT1 and NaDC3 expression and SIRT1 activity. To determine the relation between NaDC3 and SIRT1, the expression and activity of SIRT1 protein were tested after transfecting NaDC3 gene in HKC and WI38 cells. To determine the effects of NaDC3 on SIRT1 in the aging process, aging-related phenotypes were tested after transfecting sense-NaDC3 in WI38 cells, including that the SA-β-gal staining positive ratio was detected by SA-β-gal staining, cells proliferation capacity was done by MTT, cell cycle was analysesed by flowcytometry system. Moreover, above parameters were also detected in WI38 transfecting sense-NaDC3 treated with resveratrol or CR serum. To determine the pathway of NaDC3 influencing SIRT1, we detected NAD~+/NADH ratio. Meanwhile, we tested aging-related phenotypes in WI38 transfecting sense-NaDC3 treated with NAD.
Results: The results showed that the level of NaDC3 expression increased and SIRT1 expression and activity decreased in rat kidneys with aging (P<0.05). The level of SIRT1 expression and activity was significantly lower in HKC and WI38 cells transfecting sense-NaDC3 (P<0.05), while higher in HKC transfecting antisense-NaDC3 (ASNaDC3) than control cells (P<0.05). Cell prolification capacity and population doubling (PD) decreased, while the SA-β-gal staining positive ratio increased, and the cell number in G1 phase also increased in WI38 cells transfecting sense-NaDC3, compared with control group (P<0.05). But the aging-related phenotypes were reduced when WI38 cells transfecting sense-NaDC3 were treated with resveratrol or CR serum. NAD~+/NADH ratio was inhibited in WI38 cells transfecting sense-NaDC3, compared with control group (P<0.05). But when the cells transfecting sense-NaDC3 were treated with NAD~+, the aging-related phenotypes were reduced (P< 0.05).
Conclusions: Our results have demonstrated that the level of SIRT1 expression and activity decreased in kidney with aging. NaDC3 promoted senescence by inhibiting SIRT1 expression and activity, which may provide a new target of delaying and preventing renal senescence.
方法：取1日龄、3月龄、12月龄和24月龄大鼠的肾脏组织，观察NaDC3和SIRT1的表达及SIRT1的活性变化，并分离培养各时间点大鼠肾小管上皮细胞，用免疫荧光法检测SIRT1的定位和表达变化。应用逆转录病毒介导的基因转移技术，将NaDC3基因分别转移至HKC和WI38细胞内，检测不同水平的NaDC3对SIRT1表达和活性的影响。在WI38细胞转染正义NaDC3后，检测衰老的相关指标，包括用β-半乳糖苷酶活性(SA-β-gal)染色方法检测了衰老相关SA-β-gal染色阳性率，用MTT法检测了细胞增殖能力，用流式细胞仪检测了细胞周期的变化，并计数了细胞分裂的代数等。在转染正义NaDC3的同时，给与SIRT1的激动剂白黎卢醇或限食大鼠血清，观察对上述衰老相关指标的影响，以证实NaDC3是否通过SIRT1影响了衰老的进程。同时检测了NaDC3对WI38细胞NAD~+／NADH比值的影响，并给与NAD刺激转染了正义NaDC3的WI38细胞，使NAD~+／NADH比值升高，观察衰老相关的指标变化，以证实NaDC3通过降低NAD~+／NADH比值，抑制SIRT1，继而加速衰老。
结果：随大鼠月龄增长，NaDC3在肾脏的表达水平逐渐增高，而SIRT1的表达和活性均逐渐下降。SIRT1在肾小管上皮细胞核内表达，荧光强度随大鼠增龄逐渐减弱。HKC和WI38细胞转染正义NaDC3后明显抑制了SIRT1的蛋白表达和去乙酰化酶活性；HKC转染反义NaDC3后SIRT1的蛋白表达和去乙酰化酶活性有所升高。在WI38细胞转染正义NaDC3后，加速了细胞衰老，表现为细胞增殖能力下降，SA-β-gal染色阳性率明显增高，细胞分裂代数减少，停滞在G1的细胞数量增多；给与SIRT1的激动剂白黎卢醇或限食大鼠血清，则能抑制细胞出现以上衰老相关的表型。转染正义NaDC3的WI38细胞NAD~+／NADH比值降低，给与NAD刺激，提高NAD~+／NADH的比值后，细胞衰老相关的表型部分得到抑制。
结论：在大鼠肾脏中，SIRT1的表达和活性随着增龄而下降，在肾脏衰老中起重要作用。NaDC3可通过增强能量代谢而抑制NAD~+依赖的SIRT1的蛋白表达和酶活性而促进肾脏衰老。
Background and objective: Normal aging alters renal function, the high frequency of end-stage renal disease (ESRD) in the elderly. The incidence of acute renal failure is higher in older subjects, and the clinical outcome of renal failure is significantly worse with regard to mortality, requirement of dialysis, and complete recovery. So it is important to study the mechanism of aging kidney. The pathological changes of tubular-interstitial are especially important because they determine the aggravate degree of renal function. High-affinity sodium-dependent dicarboxylate cotransporter (NaDC3) is expressed in basolateral membrane of proximal renal tubular epithelial cells primarily involved in the uptake and metabolism of Krebs cycle intermediates, which include a variety of di- and tricarboxylates such as succinate, citrate, and a-ketoglutarate. It induces the aging process. The pathway of NaDC3 mediating aging would provide the targets of delaying aging. But the mechanism is yet unclear. NaDC3 provides energy for renal tubular epithelial cells, and there are tight link between energy metabolism and lifespan. SIRT1, a NAD-dependent histone deacetylase, extends many organism lifespan and reduces aging-related diseases in caloric restriction. SIRT1 links energy metabolism and lifespan through NAD. We presumed NaDC3 probably regulated lifespan through inhibiting SIRT1 function. To determine the mechanism of aging induced by NaDC3, the study designed to investigate firstly the change of SIRT1 expression and activity in kidney with aging, and then detected the effects of NaDC3 on the level of SIRT1 expression and activity in HKC and WI38 cells, senescent-related phenotypes in WI38 cells, and NAD~+/NADH ratio.
Methods: The kidneys from different month-old rats were used to investigate the change of SIRT1 and NaDC3 expression and SIRT1 activity. To determine the relation between NaDC3 and SIRT1, the expression and activity of SIRT1 protein were tested after transfecting NaDC3 gene in HKC and WI38 cells. To determine the effects of NaDC3 on SIRT1 in the aging process, aging-related phenotypes were tested after transfecting sense-NaDC3 in WI38 cells, including that the SA-β-gal staining positive ratio was detected by SA-β-gal staining, cells proliferation capacity was done by MTT, cell cycle was analysesed by flowcytometry system. Moreover, above parameters were also detected in WI38 transfecting sense-NaDC3 treated with resveratrol or CR serum. To determine the pathway of NaDC3 influencing SIRT1, we detected NAD~+/NADH ratio. Meanwhile, we tested aging-related phenotypes in WI38 transfecting sense-NaDC3 treated with NAD.
Results: The results showed that the level of NaDC3 expression increased and SIRT1 expression and activity decreased in rat kidneys with aging (P<0.05). The level of SIRT1 expression and activity was significantly lower in HKC and WI38 cells transfecting sense-NaDC3 (P<0.05), while higher in HKC transfecting antisense-NaDC3 (ASNaDC3) than control cells (P<0.05). Cell prolification capacity and population doubling (PD) decreased, while the SA-β-gal staining positive ratio increased, and the cell number in G1 phase also increased in WI38 cells transfecting sense-NaDC3, compared with control group (P<0.05). But the aging-related phenotypes were reduced when WI38 cells transfecting sense-NaDC3 were treated with resveratrol or CR serum. NAD~+/NADH ratio was inhibited in WI38 cells transfecting sense-NaDC3, compared with control group (P<0.05). But when the cells transfecting sense-NaDC3 were treated with NAD~+, the aging-related phenotypes were reduced (P< 0.05).
Conclusions: Our results have demonstrated that the level of SIRT1 expression and activity decreased in kidney with aging. NaDC3 promoted senescence by inhibiting SIRT1 expression and activity, which may provide a new target of delaying and preventing renal senescence.
引文
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2. Haim YC, Christine M, Kevin JB, et al. Calorie Restriction Promotes Mammalian Cell Survival by Inducing the SIRT1 Deacetylase. Science. 2004; 305(16): 390-392.
3. Howitz KT, Bitterman KJ, Cohen HY, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003; 425(6954):191-196.
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17. Smith JS, Brachmann CB, Celic I, et al. A phylogenetically conserved NAD~+-dependent protein deacetylase activity in the Sir2 protein family. Proc NatlAcad Sci USA. 2000; 97(12):6658-6663.
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1. Chen XM, Cao DW, Wang JZ, et al. Effects of Human Na~+/Dicarboxylate Cotransporter 3 on the Replicative Senescence of Human Embryonic Lung Diploid Fibroblasts. J GerontolA-Biol. 2005; 60A(6): 709-714.
2. Haim YC, Christine M, Kevin JB, et al. Calorie Restriction Promotes Mammalian Cell Survival by Inducing the SIRT1 Deacetylase. Science. 2004; 305(16): 390-392.
3. Howitz KT, Bitterman KJ, Cohen HY, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003; 425(6954):191-196.
4. Bela S, Erno T and Zsuzsa KV. Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures. Exp Mol Med. 2000; 32(2):88-92.
5. Cai AL, Zipfel GJ, Sheline CT. Zinc neurotoxicity is dependent on intracellular NAD levels and the sirtuin pathway. Eur J Neurosci. 2006; 24(8):2169-2176.
6. Kaeberlein M, McDonagh T, Heltweg B, et al. Substrate-specific Activation of Sirtuins by Resveratrol. J Biol Chem. 2005; 280(17):17038-17045.
7. Cohen HY, Miller C, Bitterman KJ, et al. Calorie Restriction Promotes Mammalian Cell Survival by Inducing the S1RT1 Deacetylase. Science. 2004; 305(5682):390-392.
8. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995; 92(20): 9363-9367.
9. Anderson RM, Bitterman KJ, Wood JG, et al. Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature. 2003; 423 (6936):181-185.
10. Lin SJ, Defossez PA, Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae.Science. 2000; 289(5487):2126-2128.
11. Lin SJ, Ford E, Haigis M, et al. Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev. 2004; 18(1):12-16.
12. Lin SJ, Kaeberlein M, Andalis AA, et al. Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature. 2002; 418(6895):344-348.
13. Revollo JR, Grimm AA, Imai S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian
?cells. J Biol Chem. 2004; 279(49):50754-50763.
14. Sasaki T, Maier B, Bartke A, et al. Progressive loss of SIRT1 with cell cycle withdrawal. Aging Cell. 2006; 5(5):413-422.
15. Langley E, Pearson M, Faretta M, et al. Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. EMBO J. 2002; 21(10):2383-2396.
16. Wood 3G, Rogina B, Lavu S, et al. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004; 430(7000):686-689.
17. Smith JS, Brachmann CB, Celic I, et al. A phylogenetically conserved NAD~+-dependent protein deacetylase activity in the Sir2 protein family. Proc NatlAcad Sci USA. 2000; 97(12):6658-6663.
1. McCay CM, Crowell MF. Prolonging the life span. Sci Mon. 1934; 39, 405-414.
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3. Logroscino G, Marder K, Cote L, et al. Dietary lipids and antioxidants in Parkinson's disease: a populationbased, case-control study. Ann. Neurol. 1996; 39, 89-94.
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