TNFα介导的信号在肾小球微血管和动脉大血管衰老中的作用

Kidney and vascular aging are important health problems. Chronic inflammation is implicated in the pathogenesis of kidney and vascular aging. The levels of TNFα and its soluble receptors, sTNFR1 and sTNFR2, are increased in aging. Since our preliminary data inducate that TNFα or TNFR1 deletion partly protects mice from kidney aging and furthermore, there are reports that anti-TNFα treatment reduced aging associated abnormalities in carotid artery, we postulate that TNFα ,acting via TNFR1, plays an important role in kidney ang vascular aging. NF-κB和MAPK are two down stream signal transduction pathways critically involved in TNFα meidated inflammation and cell death. Since kidney and vascular aging have been shown to be associated with the activation of NF-κB and MAPK, we further postulate that the inhibition of NF-κB or MAPK would decrease inflammatory lesions of aging kidney and reduce apoptotic cell death in aging aorta. This will be tested in aging mice with one allele of NF-κB p65 subunit deletion or a whole alleles NF-κB p50 subunit deletion. Additionally, since we have found that pentosan polysulfate (PPS), a mixture of semisynthetic polyanions that is approved by the FDA as an oral medication for interstitial cystitis, an inflammatory disease, suppresses TNFα stimulated NF-κB activation and inflammation in renal cells and reduces chronic inflammatory lesions in aging diabetic nephropathy, we propose to determine the effect of PPS on prevention and treatment of kidney and vascular aging in mice. Since PPS has an excellent long-term safety profile, the study of the effect of PPS on kidney and vascular aging may have important clinical implications. It is well known that aging kidney exhibits exacerbated inflammatory reponses to cytokine and injury, which result in increased severity of kidney aging and chronic kidney disease(s). We hypothesize that elevated oxidants act together with TNFα and other proinflammatory cytokines to increase inflammation in aging kidney. TNFα transiently activates p38 and JNK because MAPK phosphatases including MKP5 quickly dephosphorylate and inactivate p38 and JNK. However, the presence of excessive oxidative stress leads to prolonged p38 and JNK activation due to a direct modification and inactivation of phosphatase acitivity of MKP by excessive oxidants. Tnhis results in increased TNFα action and chronic inflammation in aging kidney. Thus, both the inhibition of inflammatlry cytokine(s) and oxidative stress may be required for the prevention and treatment of chronic kidney disease in aging.

慢性炎症在肾脏衰老的发生发展中起到关键的作用。我们的初步研究发现，敲出TNFα或TNFR1对肾脏衰老具有部分保护作用，而敲出TNFR2对肾脏衰老没有明显保护。由于TNFα基因敲出或抑制TNFα治疗可以减轻颈动脉衰老，我们将进一步了解TNFα及其受体对肾脏和动脉衰老的影响。然后我们研究TNFα介导的NF-κB和MAPK活化在肾脏和动脉衰老中的作用。最后，我们将探讨老年肾脏对TNFα和炎症因子反应增强的分子机制。炎症因子包括TNFα的刺激在年轻小鼠肾脏引起很短暂的炎症信号MAPK活化，因为MAPK的磷酸酶（MKP）也同时被激活。MKP通过去磷酸化的作用迅速使MAPK失活。而在老年小鼠的肾脏，由于氧化应激降低MKP磷酸酶的活性,使得MAPK不能被迅速失活，导致炎症信号延长，炎症反应增强。器官衰老的防治可能需要同时抑制炎症因子和自由基。本研究结果将有助于增加我们对肾脏和动脉衰老发病机制的认识。

炎症是造成肾脏衰老的一个重要原因。在本项目资助期间，我们重点对 TNFα，以及受体TNFR1和TNFR2在肾小球衰老发病中的作用进行探讨。结果发现，敲出TNFα和TNFR1对小鼠肾脏衰老而出现的纤维化病变具有保护作用。但只有TNFR2敲出的小鼠不发生老年所伴有的白蛋白尿。在体外培养的足突细胞用siRNA降低TNFR2表达可以减少TNFα引起的白蛋白通透性增加，而降低TNFR1表达则没有这个作用。另外，高表达TNFR2本身就可以增加白蛋白通透性。证实TNFR2可能直接参与足细胞白蛋白尿的发病。但是尽管老年TNFR2敲出小鼠不发生蛋白尿，小鼠却存在明显的肾脏纤维化，提示蛋白尿和纤维化可能不完全是一个同步的过程。从年轻（3-6月龄）及老年（24月龄）（n=6/年龄组）小鼠分离肾小球后测定p38激酶活性以及观察肾小球在接受TNFα或LPS刺激后CXCL1 mRNA表达增加，提示老年小鼠肾小球p38激酶活性增强，对TNFα和LPS刺激的炎症反应增强。体外实验证明氧自由基和碳自由基（丙酮醛）可以通过修饰MKP磷酸酶结构域的半胱氨酸和精氨酸来降低MKP磷酸酶活性，导致JNK和p38激活时间延长，从而使得TNFα等炎症因子诱导的炎症效应增强，炎症时间延长。提示老年肾小球对炎性因子反应增强且易发展为慢性炎症。为今后发展有针对性抑制TNFα介导的炎症的作用，防治肾小球衰老的进展提供预防和治疗的思路。