臺灣博碩士論文加值系統

糖尿病腎病變及末期腎炎分別佔台灣十大死因的第四及第八位。此外糖尿病腎病變在台灣是造成洗腎病患最主要的因素。早期有關腎病變的研究都著重在腎絲球及腎小管組織間隙的病變機制，包括細胞肥大與增生以及細胞外間質的增厚所導致的腎纖維化及末期腎病。近幾年來根據其他學者及我們先前的研究均發現NO、高血糖以及高度糖化終產物是造成糖尿病腎病變的三大主因。然而對於NO/PKG訊息傳遞路徑與高糖所調控細胞生物效應之間的相關性，目前為止尚未被探討過。
NO一直被認為可能與糖尿病腎病變中的腎小管組織間隙纖維化有關。不正常的高血糖所導致的腎小管組織間隙纖維化，在糖尿病腎病變中可能扮演著重要的角色。本研究中我們試圖去探討腎纖維母細胞(NRK-49F)暴露在高葡萄糖環境時，NO的表現情形及其產生的效應。首先我們在培養12、18、24小時後，發現高糖(500 mg/dl)比一般糖(100 mg/dl)明顯降低NO的合成。當加入甘露醇(400 mg/dl)使糖滲透壓提高後並無發現NO的合成量減少，因此推測上述的效應並非糖滲透壓所產生的影響。而高糖則是在處理4、8、12小時會抑制PKG蛋白質的表現。如果在高糖環境中以NO供給者與PKG活化劑刺激腎纖維母細胞，則會誘發高濃度NO表現，且iNOS與PKG蛋白質的合成量也會增加。有趣的是在刺激30分鐘後，高糖會明顯誘發JAK2與STAT1的活化，但STAT3與STAT5的活化則不受影響。在加入NO供給者及PKG活化劑後均可有效抑制上述高糖的效應。另一方面，實驗結果也證實NO供給者及PKG活化劑都能有效抑制cdk4的活化及促進p21Waf1/Cip1蛋白質(但非p27Kip1)表現。所以我們進一步推測活化NO/PKG訊息路徑能夠抑制高糖所誘發的細胞週期進行。綜合上述結果，我們認為高糖可以明顯地抑制NO生成及其訊息傳遞路徑，而透過活化NO/PKG訊息傳導方式則可藉由特定機轉來調控高糖所促進之腎纖維母細胞增生效應。

In Taiwan, diabetes mellitus and end-stage renal disease is the 4th and 8th leading cause of death, respectively. Diabetic nephropathy (DN) is the chief cause of new dialysis patients in our country. Most studies focused on the pathomechanisms of glomerular and tubulointerstitial cells in DN, which is characterized by cellular hypertrophy/hyperplasia and extracellular matrix (ECM) expansion leading to renal fibrosis and end-stage renal disease (ESRD). Based on others and our previous studies, we suggested that nitric oxide (NO), hyperglycemia and advanced glycation end products (AGEs) are three of the most significant factors in the pathogenesis of DN. However, the interactions between the signal transduction pathway of NO/cGMP-dependent protein kinase (PKG) and high glucose (HG)-mediated cellular responses remain poorly understood.
NO has been suggested to be associated with tubulointerstitial fibrosis in DN. Abnormal glucose handling in the tubulointerstitium may play an important role in the development of DN. This study was designed to investigate the effect of NO generation and action in renal fibroblasts exposed to HG. We found that HG (500 mg/dl) significantly decreased nitrite production compared with normal glucose (100 mg/dl) when the incubation period was for 12, 18, or 24 h. The inhibitory effect was not mediated by an increase in osmolarity, since raising the osmolarity by the addition of D-mannitol did not significantly decrease NO production. HG inhibited PKG expression at 4, 8, and 12 h. Both NO donors and PKG activator treatment induced high levels of NO, inducible nitric oxide synthase (iNOS), and PKG in HG-incubated cells. Interestingly, HG-induced Janus kinase 2 (JAK2)-signal transducers and activators of transcription 1 (STAT1) activation but not STAT3 or STAT5 activation at 30 min were blocked by NO donors and PKG activator. The ability of NO-PKG to inhibit HG-induced cell cycle progression was verified by the observation that NO donors and PKG activator inhibited cdk4 activation and increased p21Waf1/Cip1 (but not p27Kip1) expression in HG-treated renal fibroblasts. Collectively, our results suggest that HG significantly blunted NO signaling and activation of the NO-PKG pathway may modulate HG-enhanced mitogenic response via specific pathways.

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