Original Article
Reduction of Diabetes-Induced Oxidative Stress, Fibrotic
Cytokine Expression, and Renal Dysfunction in Protein
Kinase C–Null Mice
Yuzuru Ohshiro,
1
Ronald C. Ma,
1
Yutaka Yasuda,
1
Junko Hiraoka-Yamamoto,
1
Allen C. Clermont,
1
Keiji Isshiki,
1
Kunimasa Yagi,
1
Emi Arikawa,
1
Timothy S. Kern,
2
and George L. King
1
Diabetes induces the activation of several protein kinase C
(PKC) isoforms in the renal glomeruli. We used PKC-
/
mice to examine the action of PKC- isoforms in diabetes-
induced oxidative stress and renal injury at 8 and 24 weeks
of disease. Diabetes increased PKC activity in renal cortex
of wild-type mice and was significantly reduced (<50% of
wild-type) in diabetic PKC-
/
mice. In wild-type mice,
diabetes increased the translocation of PKC- and -1 to
the membrane, whereas only PKC- was elevated in
PKC-
/
mice. Increases in urinary isoprostane and
8-hydroxydeoxyguanosine, parameters of oxidative stress,
in diabetic PKC-
/
mice were significantly reduced com-
pared with diabetic wild-type mice. Diabetes increased
NADPH oxidase activity and the expressions of p47
phox
,
Nox2, and Nox4 mRNA levels in the renal cortex and were
unchanged in diabetic PKC-
/
mice. Increased expres-
sion of endothelin-1 (ET-1), vascular endothelial growth
factor (VEGF), transforming growth factor (TGF)-, con-
nective tissue growth factor (CTGF), and collagens IV and
VI found in diabetic wild-type mice was attenuated in
diabetic PKC-
/
mice. Diabetic PKC-
/
mice were pro-
tected from renal hypertrophy, glomerular enlargement,
and hyperfiltration observed in diabetic wild-type mice and
had less proteinuria. Lack of PKC- can protect against
diabetes-induced renal dysfunction, fibrosis, and increased
expressions of Nox2 and -4, ET-1, VEGF, TGF-, CTGF, and
oxidant production. Diabetes 55:3112–3120, 2006
D
iabetic nephropathy is characterized by glomer-
ular hyperfiltration, extracellular matrix accu-
mulation, glomerular enlargement, mesangial
expansion, and intertubular fibrosis, resulting
ultimately in diabetic glomerulosclerosis and progressive
renal insufficiency (1,2). Hyperglycemia-induced meta-
bolic and hemodynamic factors are thought to be media-
tors of this injury, which is associated with the diabetic
state (3). The hemodynamic factors implicated in the
pathogenesis of diabetic nephropathy include increased
systemic and intraglomerular pressure and activation of
various vasoactive hormone pathways including the renin-
angiotensin system and endothelins (3). This may interact
with metabolic pathways activating signaling pathways
that lead to renal injury. Multiple biochemical path-
ways have been proposed to explain the adverse effects
of hyperglycemia. Activation of diacylglycerol (DAG)–
protein kinase C (PKC) pathway (4), enhanced polyol path-
way (5), increased oxidative stress (6), and overproduction
of advanced glycation end products (AGEs) (7) have all been
proposed as potential cellular mechanisms by which hyper-
glycemia induces chronic diabetes complications.
We and others (8 –10) have previously reported that
multiple PKC isoforms are activated in each vascular
tissue of diabetic animal models, and activation of the
DAG-PKC pathway is a key mediator of diabetes vascular
complications. Immunoblotting studies have reported that
PKC- and -1 isoforms were increased in vivo in mem-
branous fractions (activated pool) of diabetic rat glomeruli
and in vitro in mesangial cells exposed to elevated glucose
levels (10), whereas PKC-2 was reported to be preferen-
tially activated in the aorta and heart of diabetic rats (8).
Whiteside and Dlugosz (11) reported that PKC- and -
isoforms were also increased in the membrane pool in the
glomeruli of diabetic rats. Treatment of diabetic animals
with a selective PKC- isoform inhibitor (LY333531 or
ruboxistaurin [RBX]) was associated with normalization
of hemodynamic changes, extracellular matrix, and histo-
logical features of glomerular damage in animal models of
diabetes (10,12,13). Phase two clinical trial results sug-
gested that RBX can decrease the loss of glomerular
filtration rate (GFR) and proteinuria in diabetic patients
already treated with inhibitors of angiotensin actions (14).
Recently, activation of NADPH oxidase and increased
reactive oxygen species (ROS) production have been
proposed as important mediators of renal dysfuncton in
diabetes (15,16). We have reported that inhibition of
PKC- by RBX can also normalize diabetes or hyperglyce-
mia-induced oxidative stress (17). PKC- has been noted
to contribute to NADPH oxidase activations in multiple
cells, including endothelial and mesangial cells (18). How-
ever, it is still unclear which of the renal abnormalities are
induced by PKC- isoforms as compared with other PKC
isoforms and which of the potential downstream biochem-
From the
1
Joslin Diabetes Center, Harvard Medical School, Boston, Massa-
chusetts; and the
2
Department of Medicine, Case Western Reserve University,
Cleveland, Ohio.
Address correspondence and reprint requests to George L. King, Research
Director, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail:
george.king@joslin.harvard.edu.
Received for publication 30 June 2006 and accepted in revised form 11
August 2006.
Y.O., R.C.M., and Y.Y. contributed equally to this work.
8-OHdG, 8-hydroxydeoxyguanosine; AGE, advanced glycation end product;
CTGF, connective tissue growth factor; DAG, diacylglycerol; ET-1, endothe-
lin-1; FF, filtration fraction; GFR, glomerular filtration rate; PAH, para-
aminohippurate; PKC, protein kinase C; RBX, ruboxistaurin; ROS, reactive
oxygen species; RPF, renal plasma flow; TGF, transforming growth factor;
VEGF, vascular endothelial growth factor.
DOI: 10.2337/db06-0895
© 2006 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
3112 DIABETES, VOL. 55, NOVEMBER 2006