果糖激酶（KHK）对糖尿病造影剂肾病的影响和机制研究

We present the hypothesis that fructokinase may have a novel role in diabetic renal disease. Fructokinase is an enzyme in fructose metabolism that is present primarily in the liver, small intestines, and proximal tubule of the kidney. Little attention has focused on the role of renal fructokinase. However, the proximal tubule may be exposed to large amounts of fructose, either via the diet, or even more so by being the site of absorption of glucose which can be converted to fructose via the aldose reductase pathway. We have preliminary evidence that in the diabetic state that fructokinase is activated in the proximal tubule, likely due to massive glucose absorption with conversion to fructose. In turn, the conversion of fructose to fructose-1-phosphate results in the production of toxic byproducts, such as uric acid and oxidants, that induce tubular injury and local inflammation. Preliminary data suggest the proximal tubular injury could have a role in acute renal disease. In this application we will test this hypothesis using fructokinase knockout mice. Specifically, we will determine the role of fructokinase in explaining the predisposition of diabetic subjects to acute kidney injury from contrast. We believe these studies are novel as they address a new mechanism for renal disease (fructokinase activation in the proximal tubule). The studies could be significant as it may provide evidence for a role for fructokinase in the mechanism of contrast-induced acute renal injury in diabetes, and could also provide a mechanism linking metabolic syndrome with acute and chronic kidney disease. Finally, our approach using fructokinase knockout mice, coupled with our preliminary data, show that the studies are feasible and achievable.

流行病学研究显示，高果糖摄入与糖尿病和慢性肾脏病密切相关。糖尿病状态下多元醇途径过度激活，导致果糖在肾脏局部的富集。果糖激酶（KHK）是果糖代谢途径的关键酶，作用产生有害代谢产物如尿酸、氧化物等，造成损伤。我们的前期研究表明①高果糖饮食可诱导产生依赖于KHK的肾小管间质损伤；②KHK在糖尿病小鼠近端肾小管高表达；③KHK基因敲除糖尿病小鼠慢性肾小管间质损伤轻于野生型糖尿病小鼠。提示KHK可能是参与糖尿病发生肾脏损伤的作用环节。我们推测，糖尿病状态下，近端小管KHK上调，是肾脏易感于各种有毒物质损伤的重要环节之一。本研究拟利用糖尿病状态下造影剂急性肾损伤作为疾病研究模型，通过KHK基因敲除鼠、KHK抑制剂、体外培养过表达和缺失表达KHK蛋白的小管上皮细胞，临床肾脏病理组织等研究手段，探讨KHK途径在糖尿病状态下急性肾损害发生环节中的重要作用，为进一步阐明糖尿病肾脏损伤的复杂机制提供理论依据。

糖尿病患者是发生造影剂肾损伤的重要易感人群。流行病学研究提示：高果糖摄入与糖尿病和慢性肾脏病密切相关。果糖激酶（KHK）是果糖代谢途径的关键酶，作用产生有害代谢产物如尿酸、氧化物等，造成损伤。我们的前期研究提示，高果糖饮食可诱导产生依赖于KHK的肾小管间质损伤；KHK在糖尿病小鼠近端肾小管高表达；KHK基因敲除糖尿病小鼠慢性肾小管间质损伤轻于野生型糖尿病小鼠。本研究推测，KHK可能是参与糖尿病发生造影剂肾脏损伤的重要环节。本研究的主要内容：①体内试验探讨KHK基因敲除糖尿病小鼠和KHK野生型糖尿病小鼠造影剂致急性肾损伤后肾脏功能及肾脏病理病变的差异；②体内试验探讨KHK抑制剂（P-CMB-3105）对糖尿病造影剂致急性肾损伤的的影响；③体外试验探讨KHK抑制剂（P-CMB-3105）对肾小管上皮细胞造影剂处理后损伤的作用；④在糖尿病造影剂肾病患者临床研究中，探讨KHK和的关系。研究主要结果和发现如下：①成功建立KHK基因敲除/野生型糖尿病小鼠造影剂致急性肾损伤模型；②KHK基因敲除糖尿病小鼠造影剂模型较野生型糖尿病小鼠造影剂模型的肾功能（Scr 及BUN）损伤情况及肾小管急性损伤（NGAL）显著改善；③AR及KHK在糖尿病小鼠，及造影剂致急性肾损伤的糖尿病小鼠肾脏中均高表达，提示KHK通过AR通路参与糖尿病鼠造影剂肾损伤的机制。④KHK基因敲除糖尿病小鼠造影剂模型较野生型糖尿病小鼠造影剂模型的炎症损伤指标（MCP-1、IL-6、TGF-β、IL-1β、和FN1等）情况显著改善；⑤造影剂致糖尿病鼠急性肾损伤模型前以不同剂量的KHK抑制剂（P-CMB-3105）预处理实验动物可部分改善糖尿病鼠的造影剂致急性肾损伤。⑥体外试验KHK抑制剂（P-CMB-3105）预处理，肾小管上皮细胞急性损伤改善，炎症NF-kB通路活化水平降低。⑦糖尿病造影剂肾病患者临床研究中提示，糖尿病造影剂肾病患者尿液中果糖总量及尿肌酐标化后果糖含量，均显著高于正常对照。本研究初步提示：KHK途径在糖尿病状态下造影剂急性肾损害发生环节中起重要作用，为进一步阐明糖尿病肾脏损伤的复杂机制提供理论依据。