NADPH oxidase-mediated upregulation of connexin43 contributes to podocyte injury

doi:10.1016/j.freeradbiomed.2012.07.012

Free Radical Biology and Medicine

Volume 53, Issue 6, 15 September 2012, Pages 1286-1297

https://doi.org/10.1016/j.freeradbiomed.2012.07.012 Get rights and content

Abstract

The gap junction protein connexin43 (Cx43) was markedly increased in podocytes in a rat model of nephrosis induced by puromycin. However, the mechanisms and roles of the altered Cx43 in podocytes are still unclear. Given that oxidative stress mediates podocyte injury under a variety of pathological situations, we examined the possible involvement of an oxidative stress-related mechanism in the regulation of Cx43. Incubation of podocytes with puromycin led to a time- and concentration-dependent loss of cell viability, which was preceded by an elevation in Cx43 levels. Concomitantly, puromycin also induced NOX4 expression and promoted superoxide (O₂⁻) generation. Inhibition of NADPH oxidase with apocynin and diphenyleneiodonium chloride or addition of the superoxide dismutase mimetic tempol completely abrogated, whereas the O₂⁻ donors menadione and 2,3-dimethoxy-1,4-naphthoquinone reproduced, the effects of puromycin on Cx43 expression and cell injury. Further analysis demonstrated that treatment of podocytes with several structurally different gap-junction inhibitors significantly attenuated the cytotoxicity of puromycin. Our results thus indicate that NADPH oxidase-mediated upregulation of Cx43 contributes to podocyte injury.

Highlights

► The mechanisms and roles of altered connexin43 (Cx43) in injured podocytes are unclear. ► Puromycin-triggered podocyte injury was preceded by increased expression of NOX4 and Cx43. ► Inhibition of NADPH oxidase abrogated the elevation in Cx43. ► Suppression of Cx43 attenuated the cytotoxic effect of puromycin.

Introduction

Podocytes are specialized cells with a complex cellular morphology and structure. They cover the outer surface of the filtering glomerular capillaries and play an important role in the maintenance of glomerular structure and function. Podocyte injury leads to proteinuria, a common feature of almost all types of glomerular diseases [1].

Podocyte injury is characterized by the rearrangement of the podocyte slit diaphragm and the actin cytoskeleton, such as nephrin, podocin, actinin α4, and CD2-associated protein [2], [3], [4], [5], [6], [7]. It is also displayed by deregulation of the activities of the TRPC6 channel [6], [7], [8]. In a rat model of nephrosis induced by puromycin aminonucleoside (PAN), we have observed a striking increase in the gap junction protein connexin43 (Cx43) in podocytes [9]. The change in Cx43 appeared to be well correlated with the initiation, progression, and regression of podocyte injury [9], [10]. However, the mechanisms responsible for the elevation in Cx43 and the role of the altered Cx43 in podocyte injury are still unclear.

One of the common mechanisms underlying podocyte injury is oxidative stress. Overproduction of reactive oxygen species (ROS) has been reported in several types of glomerular diseases [11], [12]. Therapeutic strategies against oxidative stress have been proven to be effective in prevention or attenuation of podocyte injury and proteinuria formation [13], [14].

NADPH oxidase is one of the major enzymes contributing to the production of superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) in various pathophysiological situations [11], [15]. It is composed of multiple membrane-associated and cytosolic components, including gp91phox, p22phox, p40phox, p47phox, p67phox, and Rac. Recently, various NADPH oxidase subunits have been identified in the kidney [11], [15], [16] and have been shown to participate in the regulation of renal structure and function. NADPH oxidase is also known for the regulation of multiple intracellular and plasma membrane ion channels and transporters, such as L-type channels, sarcoplasmic reticulum ATPase, the ryanodine receptor, Na⁺ channel, and K⁺ channel [15], [17]. In podocytes, NADPH oxidase has been implicated in PAN-induced expression of TRPC6 [6], [8]. Thus, it is possible that NADPH oxidase also contributes to puromycin-induced expression of Cx43 in podocytes. One of the purposes of this study was to test this hypothesis.

Gap junctions are specialized membrane regions composed of aggregates of channels that permit the direct exchange of ions, secondary messengers, and small signaling molecules among neighboring cells. Each gap-junction channel is composed of two hemichannels that reside in the plasma membrane of two closely opposed cells. The proteins that form the gap junctions are called connexins (Cxs). Up to now, more than 20 different Cx molecules have been identified. Among them, Cx43 has been extensively investigated because of its ubiquitous expression in a variety of cell types. Intercellular communication via gap junctions plays an important role in the regulation of cell functions including cell proliferation, migration, differentiation, and survival [18], [19], [20]. Gap junctions have been implicated in various pathological situations, including those associated with oxidative stress. Gap junctions, on one hand, are subjected to the regulation of oxidative stress [21], [22], [23]. On the other hand, they modulate cell responses to oxidative stress-initiated cell injury [24], [25], [26], [27]. In this context, the altered Cx43 in podocytes might affect podocyte response to oxidative stress. This possibility was also examined in this study.

Here, we present our evidence showing a critical role for NADPH oxidase and O₂⁻ in the regulation of Cx43 protein levels. In addition, we establish Cx43 as a currently unrecognized molecule contributing to oxidative stress-elicited podocyte injury.

Section snippets

Materials

A total ROS/O₂⁻ detection kit was purchased from Enzo (Tokyo, Japan). An H₂O₂ cell-based assay kit was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Annexin V–FITC apoptosis detection kit was from Bio Vision (Mountain View, CA, USA). Anti-NOX4 was from Novus Biological (Littleton, CO, USA). WST reagent was from Dojindo (Kumamoto, Japan). 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) was from Nacalai Tesque (Kyoto, Japan). Horseradish peroxidase-conjugated anti-rabbit or mouse IgG was from

NADPH oxidase underlies puromycin-induced podocyte injury

We first established the role of oxidative stress in puromycin-induced podocyte injury. For this purpose, we used puromycin hydrochloride, because it exerted a much more potent cytotoxic effect on podocytes than PAN (a well-used chemical for induction of in vivo rat podocyte injury). As shown in Figs. 1A and B, puromycin hydrochloride induced a time- and concentration-dependent loss of podocyte viability, as evaluated by WST assay. Puromycin treatment led to podocyte detachment and shrinkage of

Discussion

In this study, we demonstrated a pivotal role for NADPH oxidase and its products in the regulation of Cx43 expression in podocytes. In addition, we established Cx43 as a currently unrecognized molecule contributing to podocyte injury. Given that podocyte injury is one of the major mechanisms underlying the formation of proteinuria and progression of glomerular diseases, our findings could have important scientific and clinical implications.

NADPH oxidase is a family of enzyme complexes

Acknowledgments

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (17659255 and 20590953 to J.Y.; B21390324 to H.M.) and by a Strategic Project Grant from the University of Yamanashi.

References (58)

D. Kerjaschki
Dysfunctions of cell biological mechanisms of visceral epithelial cell (podocytes) in glomerular diseases
Kidney Int
(1994)
J. Wiggins
Podocytes and glomerular function with aging
Semin. Nephrol.
(2009)
E. Yaoita et al.
Up-regulation of connexin43 in glomerular podocytes in response to injury
Am. J. Pathol.
(2002)
S. Kinugasa et al.
Selective albuminuria via podocyte albumin transport in puromycin nephrotic rats is attenuated by an inhibitor of NADPH oxidase
Kidney Int.
(2011)
A. Akki et al.
NADPH oxidase signaling and cardiac myocyte function
J. Mol. Cell. Cardiol.
(2009)
D. Schiwek et al.
Stable expression of nephrin and localization to cell–cell contacts in novel murine podocyte cell lines
Kidney Int.
(2004)
C. Zhang et al.
Protection of podocytes from hyperhomocysteinemia-induced injury by deletion of the gp91phox gene
Free R
(2010)
adic. Biol. Med.
(2010)
S. Greiber et al.
NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate
Kidney Int.
(1998)
K. Li et al.
Reciprocal regulation between proinflammatory cytokine-induced inducible NO synthase (iNOS) and connexin43 in bladder smooth muscle cells
J. Biol. Chem.
(2011)
Y. Liu et al.
Inhibition of NADPH oxidase up-regulates connexin 43 and ameliorates electrical remodeling in rabbits with heart failure
Biomed Pharmacother
(2011)

Y.W. Zhang et al.

The gap junction-independent tumor-suppressing effect of connexin 43

J. Biol. Chem.

(2003)

J.M. Kim et al.

CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility

Science

(2003)

P. Mundel et al.

Structure and function of podocytes: an update

Anat. Embryol.

(1995)

W. Kriz et al.

The role of podocytes in the development of glomerular sclerosis

Kidney Int. Suppl.

(1994)

Z. Wang et al.

NADPH oxidase-derived ROS contributes to upregulation of TRPC6 expression in puromycin aminonucleoside-induced podocyte injury

Cell. Physiol. Biochem.

(2009)

G.I. Welsh et al.

The podocyte cytoskeleton—key to a functioning glomerulus in health and disease

Nat. Rev. Nephrol

(2012)

E.Y. Kim et al.

Insulin increases surface expression of TRPC6 channels in podocytes: role of NADPH oxidases and reactive oxygen species

Am. J. Physiol. Renal Physiol

(2012)

M. Koshikawa et al.

Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome

J. Am. Soc. Nephrol.

(2005)

P.S. Gill et al.

NADPH oxidases in the kidney

Antioxid. Redox Signaling

(2006)

J. Rincon et al.

Increased oxidative stress and apoptosis in acute puromycin aminonucleoside nephrosis

Int. J. Exp. Pathol.

(2004)

S. Oba et al.

Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant

Nephrol. Dial. Transplant.

(2008)

K. Bedard et al.

The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology

Physiol. Rev.

(2007)

M. Geiszt et al.

Identification of renox, an NAD(P)H oxidase in kidney

Proc. Natl. Acad. Sci. USA

(2000)

J. Yao et al.

Pathophysiological roles of gap junction in glomerular mesangial cells

J. Membr. Biol.

(2007)

J.C. Saez et al.

Plasma membrane channels formed by connexins: their regulation and functions

Physiol. Rev.

(2003)

J. Yao et al.

Gap junctional intercellular communication in the juxtaglomerular apparatus

Am. J. Physiol. Renal Physiol

(2009)

E.I. Azzam et al.

Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect

Oncogene

(2003)

B. Bellei et al.

Ultraviolet A induced modulation of gap junctional intercellular communication by P38 MAPK activation in human keratinocytes

Exp. Dermatol.

(2008)

V.M. Berthoud et al.

Oxidative stress, lens gap junctions, and cataracts

Antioxid. Redox Signaling

(2009)

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¹: These authors contributed equally to this work.

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Original ContributionNADPH oxidase-mediated upregulation of connexin43 contributes to podocyte injury

Abstract

Highlights

Introduction

Section snippets

Materials

NADPH oxidase underlies puromycin-induced podocyte injury

Discussion

Acknowledgments

Kidney Int

Semin. Nephrol.

Am. J. Pathol.

Kidney Int.

J. Mol. Cell. Cardiol.

Kidney Int.

Free R

adic. Biol. Med.

Kidney Int.

J. Biol. Chem.

Biomed Pharmacother

J. Biol. Chem.

CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility

Science

Structure and function of podocytes: an update

Anat. Embryol.

The role of podocytes in the development of glomerular sclerosis

Kidney Int. Suppl.

NADPH oxidase-derived ROS contributes to upregulation of TRPC6 expression in puromycin aminonucleoside-induced podocyte injury

Cell. Physiol. Biochem.

The podocyte cytoskeleton—key to a functioning glomerulus in health and disease

Nat. Rev. Nephrol

Insulin increases surface expression of TRPC6 channels in podocytes: role of NADPH oxidases and reactive oxygen species

Am. J. Physiol. Renal Physiol

Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome

J. Am. Soc. Nephrol.

NADPH oxidases in the kidney

Antioxid. Redox Signaling

Increased oxidative stress and apoptosis in acute puromycin aminonucleoside nephrosis

Int. J. Exp. Pathol.

Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant

Nephrol. Dial. Transplant.

The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology

Physiol. Rev.

Identification of renox, an NAD(P)H oxidase in kidney

Proc. Natl. Acad. Sci. USA

Pathophysiological roles of gap junction in glomerular mesangial cells

J. Membr. Biol.

Plasma membrane channels formed by connexins: their regulation and functions

Physiol. Rev.

Gap junctional intercellular communication in the juxtaglomerular apparatus

Am. J. Physiol. Renal Physiol

Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect

Oncogene

Ultraviolet A induced modulation of gap junctional intercellular communication by P38 MAPK activation in human keratinocytes

Exp. Dermatol.

Oxidative stress, lens gap junctions, and cataracts

Antioxid. Redox Signaling

Original Contribution
NADPH oxidase-mediated upregulation of connexin43 contributes to podocyte injury