醣化終產物在老年腎功能衰退進程中之角色探討

Abstract

隨著高齡社會的來臨，老人照護逐漸受到重視，而根據流行病學資料指出，有將近四成的老年人腎功能會大幅下降，更有研究指出，年長者體內具有較高濃度之醣化終產物 (advanced glycation end products, AGEs)，AGEs為還原糖與游離胺基酸或核酸經非酶反應產生的複合物，人體在正常的新陳代謝過程中即會生成AGEs，但隨著年紀增加人體清除AGEs的效率會下降，使得AGEs在體內大量堆積，目前已知AGEs的堆積與糖尿病腎病變有密切關係，亦導致腎小管纖維化及腎絲球硬化，且高濃度之AGEs將促使腎小管細胞衰老。然而AGEs對於老年腎臟功能之影響尚待釐清，因此，本研究欲探討AGEs在老年腎臟功能衰退進程所扮演之角色。首先，實驗利用6週齡、19週齡、70週齡及107週齡之C57BL/6小鼠比較不同週齡層腎臟之病理變化，並同時管餵給予70週齡小鼠AGEs抑制劑aminoguanidine (100 mg/kg)達三個月以探討AGEs在老年腎臟之影響，腎臟病理組織變化由過碘酸雪夫染色法(Periodic Acid-Schiff stain)及馬森三色染色法(Masson's trichrome stain)觀察，結果顯示70週與107週齡小鼠腎小管有明顯損傷，腎絲球出現間質擴增現象，且纖維化情形明顯增加，血液中腎病變評估指標Cystatin C顯著上升，但經過aminoguanidine處理後皆有顯著改善之情形，接著評估小鼠體內AGEs之含量，發現70週與107週齡小鼠血清中AGEs濃度皆顯著增高，在免疫組織染色(immunohistochemistry)中亦觀察到AGEs在腎臟中大量表現，同樣在餵食aminoguanidine後顯著下降，說明體內及腎臟AGEs濃度會隨著週齡增加而提升且AGEs確實促使老年期腎功能下降。為更進一步了解AGEs影響腎功能退化之機轉，利用西方墨點法觀察蛋白質表現情形，結果發現在週齡數高(70週、107週齡)的小鼠腎臟中，老化相關指標p21及p53皆大量表現，且氧化壓力指標CHOP及細胞凋亡相關指標Bax亦顯著上升，經aminoguanidine處理後顯著下降，說明年老後AGEs可能促使腎臟中氧化壓力升高，並進一步引發細胞凋亡之現象。另一方面，實驗亦發現AGEs可能會透過p38與Smad2/3訊息傳導路徑使α-SMA表現增加，最終導致腎臟纖維化。為驗證前述實驗發現，本研究亦利用D-galactose誘導老化動物模式探討AGEs在腎臟老化進程中扮演之角色，實驗利用10週齡C57BL/6小鼠並以皮下注射 (1000 mg/kg) 給予D-galactose達8週，同時以管餵小鼠aminoguanidine (100 mg/kg) 與resveratrol (40 mg/kg)，結果同樣發現小鼠血清中AGEs濃度、Cystatin C在給予D-galactose後顯著上升，經aminoguanidine與resveratrol處理後則顯著降低，而老化相關指標(p21、p53)、氧化壓力指標(CHOP)、細胞凋亡指標(Bax、Caspase-3)及纖維化指標(α-SMA)皆大量表現，經aminoguanidine與resveratrol處理後亦顯著下降，這些結果與自然老化動物模式相符，綜合上述，AGEs會促使腎臟細胞老化，並誘導氧化壓力上升，甚至進一步引起細胞凋亡與纖維化現象，最終導致老年腎臟功能衰退，但詳細機制未來仍待探討。

As the population ages, the care for the elderly become more important. It has been reported that about forty percent of the elderly had moderate to severe decline in renal function Also, levels of advanced glycation end products (AGEs) are elevated in individuals with advancing age. AGEs are a heterogeneous group of bioactive molecules formed by the nonenzymatic glycation of proteins and nucleic acids. Previous studies have reported that AGEs are highly associated with diabetic nephropathy and promote premature senescence of proximal tubular epithelial cells. However, the association between AGEs and declining renal function during aging process remains unclear. Hence, the aim of this study is to investigate the role of AGEs in decline of renal function in aging. Male C57BL/6 mice with the age of 6, 19, 70 and 107 weeks were used. To determine the effects of AGEs on aging kidney, 70-week-old mice were gavaged with aminoguanidine (Ag, 100 mg/kg), an inhibitor of AGEs, for three months. In 70- and 107-week-old mice, levels of serum Cystatin C, which is used to evaluate renal function, were significantly elevated compared with young mice. Histological examination (Periodic Acid-Schiff stain and Masson's trichrome stain) showed that mesangial expansion and renal fibrosis were increased in aging mice (70- and 107-week-old). Furthermore, deposition of AGEs was obviously increased in renal tissue of aging mice. After aminoguanidine treatment, levels of AGEs and Cystatin C were significantly decreased, and pathological changes in aging kidney were obviously improved. Western blot assay was used to investigate the mechanism for AGEs-induced renal function reduction. It was found that AGEs-induced age-related fibrosis via p38 and Smad2/3 dependent signaling pathway, and the expressions of markers ER-stress (CHOP), apoptosis (Bax) and cell senescence (p21 and p53) were all significantly increased in aging mice and then reducing after aminoguanidine treatment. Besides, to confirm the previous results, D-galactose-induced mice aging model was used. 10-week-old male C57BL/6 mice were subcutaneously injected with D-galactose (1000 mg/kg) for 8 weeks. Aminoguanidine (100 mg/kg) and resveratrol (40 mg/kg) were also administered orally at the same time. The serum AGEs levels, Cystatin C levels and the expressions of markers of fibrosis (α-SMA), ER-stress (CHOP), apoptosis (Bax and Caspase-3) and cell senescence (p21 and p53) were also significantly increased in D-galactose treated group, and significantly decreased after aminoguanidine and resveratrol treatment. These results were consistent with the findings in normal aging model. In summary, it’s demonstrated that AGEs would promote cell senescence, fibrosis, ER stress as well as apoptosis, leading to deterioration of renal function during aging process. However, further experiments are required to investigate the detailed mechanism that AGEs caused renal injury in aging kidney.