title: PKC-dependent superoxide production by the renal medullary thick ascending limb from diabetic rats.
abstract: 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.
authors.affiliation: Department of Cellular and Integrative Physiology, University of Nebraska College of Medicine, Omaha, NE 68198-5850, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Pascale H
authors.initials: PH
authors.lastname: Lane
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Pollock
authors.forename: Pamela K
authors.initials: PK
authors.lastname: Carmines
mesh_terms: D000166:Acridines; D000818:Animals; D015153:Blotting, Western; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism*; D049449:Luminescence; D049408:Luminescent Agents; D008297:Male; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*
chemical_list: D000166:Acridines; D007527:Isoenzymes; D049408:Luminescent Agents; D047428:Protein Kinase Inhibitors; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; D011493:Protein Kinase C