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

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.