Yang Jing, Lane Pascale H, Pollock Jennifer S, Carmines Pamela K
Department of Cellular and Integrative Physiology, University of Nebraska College of Medicine, Omaha, NE 68198-5850, USA.
Am J Physiol Renal Physiol. 2009 Nov;297(5):F1220-8. doi: 10.1152/ajprenal.00314.2009. Epub 2009 Sep 9.
Type 1 diabetes (T1D) is a state of oxidative stress accompanied by PKC activation in many tissues. The primary site of O2*- production by the normal rat kidney is the medullary thick ascending limb (mTAL). We hypothesized that T1D increases O2*- production by the mTAL through a PKC-dependent mechanism involving increased expression and translocation of one or more PKC isoforms. mTAL suspensions were prepared from rats with streptozotocin-induced T1D (STZ mTALs) and from normal or sham rats (normal/sham mTALs). O2*- production by STZ mTALs was fivefold higher than normal/sham mTALs (P < 0.05). PMA (30 min) mimicked the effect of T1D on O2*- production. Exposure to calphostin C or chelerythrine (PKC inhibitors), Gö6976 (PKCalpha/beta inhibitor), or rottlerin (PKCdelta inhibitor) decreased O2*- production to <20% of untreated baseline in both normal/sham and STZ mTALs. PKCbeta inhibitors had no effect. PKC activity was increased in STZ mTALs (P < 0.05 vs. normal/sham mTALs) and was unaltered by antioxidant exposure (tempol). PKCalpha protein levels were increased by 70% in STZ mTALs, with a approximately 30% increase in the fraction associated with the membrane (both P < 0.05 vs. sham). PKCbeta protein levels were elevated by 29% in STZ mTALs (P < 0.05 vs. sham) with no change in the membrane-bound fraction. Neither PKCdelta protein levels nor its membrane-bound fraction differed between groups. Thus STZ mTALs display PKC activation, upregulation of PKCalpha and PKCbeta protein levels, increased PKCalpha translocation to the membrane, and accelerated O2*- production that is eradicated by inhibition of PKCalpha or PKCdelta (but not PKCbeta). We conclude that increased PKCalpha expression and activity are primarily responsible for PKC-dependent O2*- production by the mTAL during T1D.
1型糖尿病（T1D）是一种氧化应激状态，在许多组织中伴有蛋白激酶C（PKC）激活。正常大鼠肾脏中产生超氧阴离子（O2*−）的主要部位是髓质厚壁升支（mTAL）。我们假设T1D通过一种依赖PKC的机制增加mTAL产生O2*−，该机制涉及一种或多种PKC亚型的表达增加和转位。从链脲佐菌素诱导的T1D大鼠（STZ mTALs）以及正常或假手术大鼠（正常/假手术mTALs）制备mTAL悬浮液。STZ mTALs产生O2*−的量比正常/假手术mTALs高五倍（P < 0.05）。佛波醇酯（PMA，30分钟）模拟了T1D对O2*−产生的影响。在正常/假手术和STZ mTALs中，暴露于钙泊三醇C或白屈菜红碱（PKC抑制剂）、Gö6976（PKCalpha/beta抑制剂）或罗特lerin（PKCdelta抑制剂）可使O2*−产生量降至未处理基线的<20%。PKCbeta抑制剂无作用。STZ mTALs中的PKC活性增加（与正常/假手术mTALs相比，P < 0.05），且抗氧化剂暴露（tempol）对其无影响。STZ mTALs中PKCalpha蛋白水平增加70%，与膜相关的部分增加约30%（两者与假手术相比，P < 0.05）。STZ mTALs中PKCbeta蛋白水平升高29%（与假手术相比，P < 0.05），膜结合部分无变化。各组之间PKCdelta蛋白水平及其膜结合部分均无差异。因此，STZ mTALs表现出PKC激活、PKCalpha和PKCbeta蛋白水平上调、PKCalpha向膜的转位增加以及O2*−产生加速，而抑制PKCalpha或PKCdelta（但不是PKCbeta）可消除这种情况。我们得出结论，PKCalpha表达和活性增加主要是T1D期间mTAL依赖PKC产生O2*−的原因。
