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Stra13, a prostaglandin E₂-induced gene, regulates the cellular redox state of podocytes ¹

MARTIN J. BEK^*, STEPHANIE WAHLE^*, BARBARA MÜLLER^*, THOMAS BENZING^*, TOBIAS B. HUBER^*, MATTHIAS KRETZLER, CLEMEN COHEN, ANDREA BUSSE-GRAWITZ^* and HERMANN PAVENSTÄDT^*,2

^* Department of Medicine, Division of Nephrology, Freiburg University Hospital, Freiburg, Germany; and
Medizinische Poliklinik, Ludwig-Maximilians-University of Munich, Munich, Germany

²Correspondence: Freiburg University Clinics, Department of Internal Medicine, Division of Nephrology, Hugstetter Str. 55, D-79106 Freiburg, Germany. E-mail: paven{at}med1.ukl.uni-freiburg.de

SPECIFIC AIMS

Radical oxygen species (ROS) cause severe damage of podocytes and thus play a detrimental role in the development of several forms of glomerular disease. In this study, we investigated whether Stra13, a gene induced by retinoic acid and prostaglandin E₂ (PGE₂), influences the cellular redox state of podocytes and plays a protective role in ROS-induced cell damage.

PRINCIPAL FINDINGS

1. Stra13, which has been recently identified as a retinoic acid-inducible gene of the basic helix–loop–helix family, is differentially induced by PGE₂ in podocytes
Likewise, forskolin and retinoic acid induce an increase of Stra13 mRNA expression. PGE₂ causes a short-term induction of Stra13 protein with a maximal expression at 1 h, whereas retinoic acid causes a longer induction of Stra13 protein with an expression for up to 4 h. Immunohistochemical studies show that Stra13 is mainly expressed in tubular cells. After perfusion of mouse kidneys with PGE₂, an increased expression of Stra13 was found in tubular and glomerular cells, including podocytes.

2. To study the role of Stra13 in podocyte function, Stra13 was stably overexpressed in mouse podocytes
Stra13 overexpressing podocytes show an 10-fold decrease in reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide anion generation (Fig. 1 ). Like Stra13 overexpressing cells, PGE₂ and retinoic acid cause a time-dependent decrease of NADPH-dependent superoxide anion generation in untransfected podocytes. To prove that Stra13 is responsible for the effects of retinoic acid on NADPH-oxidase activity, antisense experiments with Stra13 antisense oligonucleotides (AS) were performed. Preincubation with control AS did not influence retinoic acid-induced inhibition of NADPH-oxidase activity. In contrast, preincubation with AS partially reversed retinoic acid-induced inhibition of NADPH-oxidase activity.

View larger version (35K): [in this window] [in a new window]   Figure 1. (Upper panels) Western blot experiments showing Stra13 protein expression in mouse podocytes overexpressing Stra13 and control cells transfected with vector only (Co). There is an 60% increase in Stra13 expression (n=4; values are means±SEM;*, P<0.05 vs. unstimulated controls, t-test). (Left lower panel) Effect of reduced nicotinamide adenine dinucleotide (NADH; 0.1 mM) and NADPH (0.1 mM) on O2– production in Stra13 overexpressing podocytes and control cells. Addition of NADH did not stimulate the NADH/NADPH oxidoreductase enzyme complex in podocytes, neither in podocytes overexpressing Stra13 nor in control cells. Addition of NADPH (0.1 mM) increased O2– production in control cells. This effect was 10-fold decreased in Stra13 overexpressing podocytes. (Right lower panel) Summary of total O2– generation during the experimental period of 15 min (areas under the curves). There was a significant decrease in total O2– generation in Stra13 overexpressing podocytes in response to NADPH stimulation compared with control cells (n=4–5; values are means±SEM; *, P<0.05 controls, t-test).

3. cDNA expression analysis shows that mRNA expression for heme oxygenase 1 (HO-1), a protein with important antioxidative and anti-inflammatory properties, is markedly increased in Stra13 overexpressing podocytes
Western blot analyses confirm that HO-1 protein expression is increased in Stra13 overexpressing podocytes, an effect that can be mimicked by retinoic acid.

4. To prove possible cytoprotective properties of Stra13 against oxidative stress, Stra13 overexpressing podocytes and control cells were stimulated with ROS
Stra13 overexpressing podocytes show a markedly reduced toxic lactate dehydrogenase (LDH) release and cell death rate after stimulation with ROS (Fig. 2 ). To prove that the observed benefit under oxidative stress in Stra13 overexpressing cells can directly be linked to HO-1 production, we preincubated Stra13 overexpressing cells and control cells with zinc protoporphyrin IX (ZnPP; 10 µM), an inhibitor of HO-1 induction, and deferroxamine (200 µM), a potent inhibitor of lipid peroxidation. Preincubation with ZnPP reversed the protective effect of Stra13 overexpression so that LDH release in control cells and Stra13 overexpressing cells was not different any more. Preincubation with deferroxamine did not influence the protective properties of Stra13 overexpression.

View larger version (50K): [in this window] [in a new window]   Figure 2. (Left panel) LDH release in podocytes overexpressing Stra13 and control cells after 4 h of stimulation with xanthine/xanthine oxidase (X/XO) or vehicle (Co). There is a significant decrease in toxic LDH release in Stra13 overexpressing cells compared with control cells (n=6/12; values are means±SEM; *, P<0.05 controls, t-test). (Right panels) Cell morphology of podocytes overexpressing Stra13 and control cells stimulated with high concentrations of X/XO (50 µM/50 mU/ml) or vehicle (H2O). Pictures were taken of cells before analysis of LDH release. In accordance with the data shown in the upper panel, cell damage (rounded cells) is visible in control cells stimulated with X/XO but not in Stra13 overexpressing cells.

CONCLUSION AND SIGNIFICANCE

The helix–loop–helix protein Stra13 has been shown to induce differentiation in neuronal cells. Recent studies suggest that Stra13 is a key regulator of lymphocyte activation, which is important for maintenance of self-tolerance and for constraint of autoimmunity. Stra13-deficient mice exhibit defects in several phases of CD4⁺ T cell activation and develop an autoimmune disease and an immune-complex glomerulonephritis. ROS production has been implicated in the pathogenesis of several diseases, such as cancer, diabetes mellitus, atherosclerosis, ischemia, and glomerulonephritis. Furthermore, HO-1 is known to mediate antioxidant cytoprotective effects and also provide cytoprotection by facilitating iron extrusion. Our data demonstrate that Stra13 is a potent inhibitor of superoxide anion generation, regulates the expression of HO-1 in podocytes, and thus provides an important link amongst Stra13 expression, cellular redox state, and cytoprotection (Fig. 3 ). The Stra13-inducing agonists retinoic acid and PGE₂ have been implicated as inhibitors of glomerular injury; for example, retinoic acid reduces glomerular injury and albuminuria in animal models of glomerular injury. Activation of the NADPH oxidase leading to generation of ROS has been shown to play a major role in the pathogenesis of proteinuria. Our data show a new link between the actions of PGE₂ and retinoic acid and inhibition of oxidative stress and cell injury in podocytes. Further work will be necessary to characterize the molecular pathway between Stra13 and proteins that regulate the enzymatic activity of the NADPH-oxidase complex.

View larger version (20K): [in this window] [in a new window]   Figure 3. Summary of the effects of Stra13 on the regulation of NADPH oxidase and HO-1. SOD, Superoxide dismutase.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0250fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0250fje

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