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This Article Full Text Full Text (PDF) All Versions of this Article: 297/5/F1220    most recent 00314.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Yang, J. Articles by Carmines, P. K PubMed PubMed Citation Articles by Yang, J. Articles by Carmines, P. K

PKC-dependent superoxide production by the renal medullary thick ascending limb from diabetic rats

Departments of ¹ Cellular and Integrative Physiology and ; ²Pediatrics, University of Nebraska College of Medicine, Omaha, Nebraska; and ; ³Vascular Biology Center, Medical College of Georgia, Augusta, Georgia

Submitted June 2, 2009 ; accepted in final form September 8, 2009

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 (PKC/β inhibitor), or rottlerin (PKC inhibitor) decreased O₂^•– production to <20% of untreated baseline in both normal/sham and STZ mTALs. PKCβ 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). PKC protein levels were increased by 70% in STZ mTALs, with a 30% increase in the fraction associated with the membrane (both P < 0.05 vs. sham). PKCβ protein levels were elevated by 29% in STZ mTALs (P < 0.05 vs. sham) with no change in the membrane-bound fraction. Neither PKC protein levels nor its membrane-bound fraction differed between groups. Thus STZ mTALs display PKC activation, upregulation of PKC and PKCβ protein levels, increased PKC translocation to the membrane, and accelerated O₂^•– production that is eradicated by inhibition of PKC or PKC (but not PKCβ). We conclude that increased PKC expression and activity are primarily responsible for PKC-dependent O₂^•– production by the mTAL during T1D.

diabetes mellitus; rottlerin; calphostin C; chelerythrine; Gö6976