Supplementary Material for: Evaluation of JNK Blockade as an Early Intervention Treatment for Type 1 Diabetic Nephropathy in Hypertensive Rats

<i>Background/Aims:</i> The c-Jun amino-terminal kinase (JNK) signaling pathway is activated in human kidney diseases and promotes renal injury in experimental glomerulonephritis. In this study, we examined whether JNK signaling plays a role in the development of diabetic nephropathy or in regulating hypertension, which exacerbates diabetic renal injury. <i>Methods:</i> Diabetes was induced in spontaneously hypertensive rats (SHR) using streptozotocin. At week 16 of diabetes, rats with equivalent hyperglycemia and albuminuria were randomized into groups which received no treatment, vehicle alone or a selective JNK inhibitor (CC-930, 60 mg/kg/bid) for 10 weeks. These rats were assessed for hypertension and progression of renal damage. <i>Results:</i> At week 16, diabetic rats showed increased kidney JNK activation compared with nondiabetic controls. Effective JNK inhibition was demonstrated at week 26 by reductions in c-Jun phosphorylation. CC-930 did not affect blood pressure, kidney hypertrophy, glomerular hyperfiltration, podocyte loss, glomerular fibrosis or tubulointerstitial injury in diabetic SHR. However, CC-930 reduced macrophages and <i>ccl2</i> mRNA levels in diabetic kidneys. In contrast, CC-930 exacerbated albuminuria at week 26, which was associated with reduced glomerular mRNA levels of the podocyte-specific molecules, nephrin and podocin. <i>Conclusion:</i> JNK inhibition does not prevent the progression of early diabetic renal injury in hypertensive rats, which contrasts with the ability of JNK inhibition to suppress albuminuria and injury in experimental glomerulonephritis.

<i>背景与目的：</i> c-Jun氨基末端激酶（c-Jun amino-terminal kinase，JNK）信号通路在人类肾脏疾病中被激活，且在实验性肾小球肾炎模型中可介导肾损伤进展。本研究旨在明确JNK信号通路是否参与糖尿病肾病的发生发展，或在调控可加重糖尿病性肾损伤的高血压状态过程中发挥作用。<i>方法：</i> 本研究通过链脲佐菌素（streptozotocin）诱导自发性高血压大鼠（spontaneously hypertensive rats，SHR）建立糖尿病模型。糖尿病造模第16周时，选取血糖水平与白蛋白尿程度匹配的大鼠随机分为三组：空白对照组、仅赋形剂处理组，以及选择性JNK抑制剂CC-930（60 mg/kg，每日两次）给药组，干预周期为10周。随后对各组大鼠的高血压状态与肾损伤进展情况进行系统性评估。<i>结果：</i> 糖尿病造模第16周时，糖尿病大鼠的肾脏JNK激活水平显著高于非糖尿病对照组。造模第26周时，通过检测c-Jun磷酸化水平显著降低，证实JNK抑制效果确切。CC-930给药对糖尿病SHR的血压、肾脏肥大、肾小球高滤过、足细胞丢失、肾小球纤维化及肾小管间质损伤均无显著改善作用。然而，CC-930可降低糖尿病大鼠肾脏内的巨噬细胞浸润及ccl2 mRNA表达水平。与之相反，造模第26周时CC-930可加重大鼠白蛋白尿，该现象与足细胞特异性分子肾病蛋白（nephrin）及足孔蛋白（podocin）的肾小球mRNA水平降低密切相关。<i>结论：</i> 在高血压大鼠模型中，JNK抑制无法阻断早期糖尿病性肾损伤的进展，这与JNK抑制在实验性肾小球肾炎中可减轻白蛋白尿与肾损伤的作用效果截然相反。