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Activation of Protein Kinase C Isozymes Protects LLCPK1 Cells

Posted on: Thursday, 20 November 2003, 06:00 CST

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Original Report: Laboratory InvestigationAmerican Journal ofNephrology Am J Nephrol 2003;23:380389

DOI: 10.1159/000073984Activation of Protein Kinase C Isozymes

Protects LLCPK1 Cells from H2O2 Induced

Necrotic Cell DeathDina Polosukhina Kurinji Singaravelu Babu J. PadanilamDepartment of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebr., USAReceived: May 21, 2003

Accepted: August 29, 2003

Published online: October 9, 2003Key WordsAcute renal failure W Proximal tubular cells W Necrosis WOxidant injury W Cell deathAbstractBackground/Aims: We have previously reported that

ischemia/reperfusion injury (IRI) to the kidney leads to

induced expression of RACK1 and changes in the level of

expression and subcellular distribution of PKC isozymes, II and . In order to further define the role of PKC isozymes in IRI we investigated the effect of activation or

inhibition of the isozymes on cytotoxicity mediated by

H2O2 in LLCPK1 cells. Methods: Cytotoxicity was analyzed

by Trypan blue assay and LDH release assay. Translocation of PKC isozymes postinjury in LLCPK1 cells was analyzed by immunostaining and Western blot analysis. Results: Western blot analysis showed that the expression

of PKC- was up-regulated in a triphasic pattern with the

initial induction within the first 10 min of injury followed

by higher levels of expression at 2 and 24 h postinjury.

The expression of PKC- was highly induced within the

first 15 min of injury but its expression was down-regulated to that of normal levels by 30 min postinjury. Immunocytochemistry showed that both PKC- and PKC-

translocated to the nucleus and perinuclear region during

H2O2 treatment. Following injury, PKC- expression was

localized to the nuclear membrane at earlier time points

but a translocation to the nucleus occurred at later time

points. PKC- translocated to nucleus at 30 minutes post

injury and relocated back to the nuclear membrane at later time points. Conclusion: These data suggest that activation of PKC- and PKC- is involved in the H2O2 induced

injury of LLCPK1 cells.Copyright 2003 S. Karger AG, BaselIntroductionReperfusion of the kidney after an ischemic incident

results in damage to the S3 segments of the proximal

tubule located in the outer medulla and inner cortex [1].

The pathophysiological consequences during the tissue

repair and regeneration phase depend on molecular responses mediated by various signal transduction pathways [2, 3].Protein kinase C (PKC) is a family of proteins involved

in a multitude of signal transduction pathways regulating

various adaptive responses of the cell including homeostasis, migration, proliferation, apoptosis, endothelial

function, remodeling of the actin cytoskeleton, membrane

permeability and modulation of ion channels [4].The expression of the PKC isozymes , I, II, and

isoforms has been characterized in the kidney [5]. PKC

has previously been shown to be induced in cardiac, neuronal and skeletal ischemia. Cardiac ischemia is found to

induce the expression and activation of PKC isozymes ,

, and postinjury [6, 7]. Recent data demonstrate that

different isozymes may have protective or damaging roles

after cardiac ischemia both in vitro and in vivo. It was

shown that inhibiting PKC or activating -PKC protected cardiac tissues from simulated ischemic injuryABCFax + 41 61 306 12 34E- Mail karger@karger.ch

www.karger.com

2003 S. Karger AG, BaselAccessible online at:

www.karger.com/ajnBabu J. Padanilam, PhDDepartment of Physiology and Biophysics

984575 Nebraska Medical Center

Omaha, NE 68198-4575 (USA)

Tel. +1 402 559 3575, Fax +1 402 559 4438, E-Mail bpadanilam@unmc.eduwhile activation of both isozymes induced nonpathological hypertrophy of the heart [6].We have previously reported that the expression of the

receptor for activated C kinase (RACK 1) is induced after ischemia/reperfusion injury to the kidney [8]. This

prompted us to determine if the induced expression of

RACK1 is accompanied by the enhanced expression of

PKC isozymes. The expression of the PKC isozymes ,

II and are induced and activated after IRI in rat kidneys [9]. The spatial and temporal expression pattern of

the isozymes suggested that they may be involved in both

the injury and/or regeneration process. The mechanisms

by which the activation of the PKC isozymes may alter

the outcome after IRI are largely unknown.In order to determine the role of PKC isozymes after

IRI in the kidney, we studied if the inhibition or activation of the isozymes alters the outcome in an in vitro model of the injury. Since superoxides play a significant role

after IRI, oxidant injury induced by H2O2 in cell culture

models has been widely used as an in vitro model for the

injury [10, 11]. Our results demonstrate that PKC isozymes are induced and translocated after oxidant injury

in LLCPK1 cells. Further, activation of PKC isozymes

protected LLCPK1 cells from H2O2 induced oxidant

injury.Materials and MethodsCell CultureThe porcine-derived renal proximal tubular cell line LLCPK1

(ATCC, Rockville, Md., USA) were cultured in Dulbeccos modified

Eagles medium (DMEM) supplemented with 5% fetal bovine serum, 5% bovine calf serum, 2 mM glutamine, 100 U penicillin G/ml

and 100 g streptomycin/ml. Experiments were performed on confluent monolayer cultures.H2O2 Mediated Oxidant InjuryOxidant injury to confluent monolayer cultures was induced by

adding H2O2 to a final concentration of 500 M to the medium and

incubating for 30 min. After the injury, the cultures were rinsed free

of the injury medium by washing 3 times with phosphate-buffered

saline (PBS). Supplemented medium was then added to the cultures

and incubated for various recovery time periods.Treatment with PKC Inhibitors and ActivatorsConfluent monolayer of LLCPK1 cells grown on 6-well plates

were pretreated with PKC activators PMA (1 M ), bryostatin

(100 nM), 1,2-dioctanoyl-sn-glycerol (DOG) (1 M ), 1-oleoyl-2- ace-

tyl-sn-glycerol (OAG) (1 M) or pan-specific PKC inhibitors Calphostin C (500 nM), GF 109203X (200 nM) for 30 min in DMEM

before the injury. Inhibition of PKC activity also was done by prolonged incubation in presence of 1 M PMA. Stock solutions of each

of activators and inhibitors were prepared in dimethyl sulfoxide

(DMSO). DMSO also was added to control cultures to a final concentration equivalent to that in the treated cultures. The same concentration of the activators and incubators were included in the media

during and after oxidant injury.Trypan Blue Exclusion StudiesThe number of cells undergoing necrotic cell death was quantitated using the Trypan blue exclusion assay as previously described[10].LDH Release AssayLDH release assay was done using the cytotoxicity detection kit

(Roche Molecular Biochemicals, Indanapolis, Ind., USA) according

to the manufacturer.Western Blot AnalysisControl cells and cells treated with H2O2 were collected and the

protein was isolated as previously described [12]. Western blot analysis was performed using individual PKC isozyme specific antibody

and chemiluminescent detection was performed using the ECL

detection kit (Amersham-Pharmacia Biotech, Inc., Piscataway, N.J.,

USA). The intensity of each band was quantified using Lab Works

software (UVP Bioimaging systems, Upland, Calif., USA). The level

of expression for each isozyme was quantified using at least three

immunoblots from separate experiments.Extraction of Nuclear and Cytoplasmic FractionsControl cells and cells treated with 500 M H2O2 were collected

at various time points during and after injury. The nuclear extract

and cytoplasmic fractions were separated using the NE-PER Nuclear

and Cytoplasmic extraction reagents (Pierce, Rockford, Ill., USA)

according to the manufacturer. 50 g of protein from the cytoplasmic

and nuclear fractions derived from various time points were run on

an acrylamide gel and immunoblotted to PKC- or PKC- antibodies

as described above.Apoptosis DetectionControl cells and cells treated with H2O2 were collected at various

time points postinjury and centrifuged at 1,000 rpm for 5 min. The

cells were then fixed in PBS containing 4% formalin for 10 min and

stained with Hoechst 33342 (500 ng/ml) in PBS containing 80% glycerol. A small aliquot of the cell suspension was then transferred to a

microscopic slide and cover slipped. The nuclear morphology of the

cells was analyzed using a Leica DMR fluorescent microscope.Immunofluorescence MicroscopyLLCPK1 cells plated on chamber glass slides (Lab-Tek II chamber

slide systems, Fisher Scientific) were injured using 500 M H2O2. At

different time points post-injury, the cells were fixed with methanolacetone (1:1) for 10 min at 20 C, and were then air-dried. The cells

were incubated with TBST containing 10% calf serum for 10 min at

room temperature, incubated with primary antibodies for 1 h at

37 C, and washed three times for 5 min each with PBS. The cells

were incubated for 1 h at 37 C with secondary antibodies (Cy3- conjugated goat anti-rabbit or Cy3-conjugated goat anti-mouse antibodies). The cells were washed three times for 5 min each with PBS

and mounted in PPD media (90% glycerol, 0.1! PBS and 1 mg/ml

p-phenylenediamine). Samples were viewed using a Leica DMR fluorescent microscope and the images were captured with an Optronics

digital camera. Images were processed using Adobe Photoshop software for final layout.PKC in Renal Oxidant Injury Am J Nephrol 2003;23:380389 381Statistics The data are given as mean values B SE. The paired Students t

test was used to compare mean values within one experimental

group. One way ANOVA with post test was used to compare mean

values from two groups. p ! 0.05 was considered statistically significant.Materials The primary rabbit polyclonal antibodies for all of the PKC isozymes and -actin were purchased from Santa Cruz Biotechnology,

Inc. (Santa Cruz, Calif., USA). PMA, OAG, DOG, Calphostin C, and

GF 109203X were purchased from BioMol Research Laboratories,

(Plymouth Meeting, Pa., USA). H2O2 and Trypan Blue dye were purchased from Sigma Chemical Co. St. Louis, Mo., USA.ResultsEffect of PKC Activators and Inhibitors on Cellular Injury

Confluent monolayer of LLCPK1 cells were subjected

to oxidant injury using 500 M H2O2. In order to determine the effect of PKC activation on necrotic cell death,

the cells were pretreated with the PKC activators PMA,

OAG, DOG or bryostatin. The effect of the inhibitors was

determined using the pan-specific PKC inhibitors Calphostin C and GF 109203X as described in methods.

Inhibition of PKC activity also was done by overnight

exposure of LLCPK1 cells to 1 M PMA. The number of

cells undergoing necrotic cell death was expressed as a

percentage of the total number of cells in the field. As

shown in figure 1, H2O2 induced 21.43, 22.38 and 39.9%

necrotic cell death at 1, 2 and 3 h postinjury, respectively.

Incubation of the cells in presence of PKC activators significantly (n = 12, p ! 0.0001) reduced necrotic cell death

at all three time points examined. In order to determine if

any of the cells are undergoing apoptotic form of cell

death following 500 M H2O2 treatment, injured cells and

untreated cells were stained with Hoechst 33342 nuclear

stain. Morphological changes in the nuclei were assessed

using fluorescent microscopy. No variation in the number

of cells undergoing apoptosis was observed between

treated and untreated cells at any of the time points studied (data not shown). Inhibition of PKC activity using

pan-specific inhibitors or by prolonged exposure to PMA

had no effect on the cellular injury at 1 and 2 h postinjury

and the results are shown in figure 2. Activation or inhibition of PKC isozymes had no significant effect on cell

death at any time during the 30-min injury period (data

not shown).In order to examine the effects of PKC inhibition and

activation on H2O2-induced plasma membrane damage,

LDH release into the extracellular fluid was measured as aFig. 1. Effect of activation of PKC isozymes on necrotic cell death.

LLCPK1 cells were injured by exposing to 500 M H2O2 for 30 min

and the cells were allowed to recover to various time periods. Necrotic cell death was analyzed by Trypan blue exclusion studies. Data are

means B SE. Data for each time point was derived from 12 separate

experiments. * Denotes a significant difference (p ! 0.0001; n = 12)

in cell death between H2O2 treated group and cells treated with H2O2

in presence of PKC activators. Statistical analysis was done using

ANOVA followed by the Turkey-Kramer multiple comparisons test.Fig. 2. Effect of inhibition of PKC isozymes on necrotic cell death.

The cellular injury and data analyses were performed as described in

the legend to figure 1.382 Am J Nephrol 2003;23:380389 Polosukhina/Singaravelu/PadanilamFig. 3. Effect of the PKC activators PMA orOAG on H2O2 induced LDH release. LLCPK1

cells were exposed to 30 min of 500 M H2O2

injury in presence or absence of 1 M PMA or

1 M OAG and the amount of LDH released

was measured at the indicated time points. Control represents LDH release from cells that were

incubated in DMEM in the absence of H2O2 and

other agents. Results are means B SE for 4

experiments. * Denotes a significant difference

(p ! 0.005) in cell death between H2O2 treated

group and cells treated with H2O2 in presence of

PKC activators. Data analysis was done as described in figure 1.C 0 2 10 15 30 1 h 2 h 3 h 1 dazb-actinFig. 4. Western blot analysis of protein derived from LLCPK1 cells

that were cultured in DMEM only (control), and LLCPK1 cells that

underwent 30 min of 500 M H2O2 mediated injury followed by various recovery periods in DMEM. 50 g of protein extracted from the

cells was resolved on 420% polyacrylamide gels, transferred to ECL

membranes and immunoblotted with PKC- or - specific antibodies. A separate gel containing the same amount of protein in each

lane was immunoblotted with -actin antibody to confirm equal

loading.marker of lethal cell injury (fig. 3). Confluent monolayer

of LLCPK1 cells were exposed to 500 M H2O2 for 30 min

in the presence and absence of the PKC activator PMA or

OAG. Exposure of the cells to H2O2 increased LDH

release from 3.36 B 0.34 to 14.5 B 1.85% at 1 h, to 39 B1.21% at 2 h and to 62.5 B 2.5% at 3 h (p ! 0.005 vs.

control, n = 4). LDH release in the extracellular fluid was

significantly reduced by pre-treatment of the cells with the

PKC activators PMA or OAG at all three time points

studied. PKC activation using PMA led to significant

decrease in LDH release at 1 h postinjury to 7.7 B 1.85%,

to 25.3 B 4.85% at 2 h and to 38.5 B 7.98% at 3 h postinjury (p ! 0.005, n = 4). PKC inhibition using pan-specific

PKC inhibitors did not reduce LDH release at any of the

three time points. No significant change in the LDH

release was observed during the 30 min of injury or at

0 min postinjury.PKC Expression in LLCPK1 Cells following Oxidant

InjuryThe time course of expression of PKC isozymes in

LLCPK1 cells during oxidant injury was determined by

Western blotting. We have previously shown that PKC-,

II, and are the isozymes induced in kidneys after renal

ischemia. Based on this finding, we focused on the expression pattern of these three isozymes in oxidant-injured

LLCPK1 cells. Whole cell extracts from injured and control cells were size-fractionated on SDS-poly-acrylamide

gels and transferred to nylon membranes followed by

immunoblotting with PKC- and antibodies. As shown

in figure 4, the expression of PKC- was induced within

the first 10 min postinjury and its expression was downregulated to normal levels by 30 min postinjury. Its

expression was further upregulated at 1 h and reached a

second peak at 2 h postinjury. Its expression was downregulated at 3 h and upregulated at 1 day postinjury. This

triphasic pattern of expression of PKC- was similar to

that we observed in vivo [9]. The level of expression of

PKC- was highly induced during the first 15 min of injury compared to the control levels but its level of expression was down- regulated to normal levels at 30 min postinjury. Unlike PKC-, the level of expression of PKC-

was not upregulated at later time points postinjury (fig. 4).

Quantitation of the data from 3 immunoblots showedPKC in Renal Oxidant Injury Am J Nephrol 2003;23:380389 383Fig. 5. Quantitation of the expression level ofPKC- and - after H2O2 injury. 50 g of total

protein lysates from various time points derived

from cells that underwent injury as described in

figure 4 were immunoblotted to PKC- and -

specific antibodies. The intensity of the bands

was quantitated as described in Methods. Data

for each time point was derived from three separate experiments. Data are means B SE. * Denotes a significant difference (p ! 0.05) in the

expression of PKC- or - between untreated

cells (control) and H2O2 injured cells at various

time points as analyzed by Students t test.that the change in the level of expression of PKC- was

significant at 2 min, 10 min, 2 h and 1 day time points as

shown in figure 5. The level of expression of PKC- was

significantly higher at 2 min, 10 min and 15 min of injury

but was not increased at 30 min, 1 h and later time points

(fig. 5). No change in the level of expression of PKC-II

was observed at any of the time points.Subcellular Localization of PKC Isozymes in LLCPK1

Cells following Oxidant InjuryTo analyze the subcellular localization of PKC- and

PKC- in LLCPK1 cells during and after H2O2 injury, we

examined the distribution of the isozymes at various time

points by immunofluorescence microscopy. In control

cells, PKC- is distributed homogeneously in the cytoplasm with a higher concentration in the perinuclear

region (fig. 6A). The cells were exposed to 500 M H2O2

and the translocation of the PKC- isozyme was examined at 2 min during the exposure. The intensity of PKC-

staining is increased and it is translocated almost entirely

to the perinuclear region (fig. 6B). At 5 min during the

exposure, PKC- isozyme is translocated to the perinuclear region as well as into the nuclei of several cells

(fig. 6C). At 10 min during the exposure to H2O2, the

PKC- isozyme is completely translocated to the nuclei

with a concomitant decrease in the cytoplasm (fig. 6D). In

order to clearly demonstrate the perinuclear and nuclear

translocation of PKC- at 2, 5 and 10 min during the injury, high magnification photographs of individual cells

representing these time points are shown in figure 6F, G

and H, respectively. Localization of PKC- in control

cells at higher magnification is shown in figure 6E. The

cells are also stained with the nuclear dye Hoechst 33258

to localize the nucleus.In order to study the expression and translocation of

the PKC- isozyme during the recovery period, cells that

were injured by exposing to H2O2 for 30 min were washed

free of H2O2. The cells were allowed to recover in the normal media for various periods of time. Exposure of the

cells to H2O2 for 30 min caused the cells to round up. At 2,

5 and 10 min postinjury, a relocalization of the PKC-

isozyme from the nucleus to the perinuclear region is

observed and is shown in figure 6H, I and J, respectively.The translocation of PKC- from the cytoplasm to the

nucleus was confirmed by western blotting. Cytoplasmic

and nuclear extracts from LLCPK1 cells that underwent

H2O2 injury at various time points during and postinjury

were isolated and immunoblotted to PKC- antibody. As

shown in figure 7, PKC- expression is observed both in the

cytoplasmic and nuclear fractions at 5 min during the injury while at 10 min, majority of the expression is observed in

the nuclear fraction. At 2 and 10 min postinjury, PKC-

expression is limited to the cytoplasmic fraction.In control cells, the expression of PKC- is localized to

the cytoplasm and weakly along the plasma membrane

(fig. 8A). At 2 min during the exposure to H2O2, the

enzyme is primarily located to the nuclear membrane in

most of the cells and within the nuclei of a few cells

(fig. 8B). At 5 min during the injury the PKC- isozyme

translocated to the nuclei of majority of the cells (fig. 8C).

At 10 min during the injury, the isozyme completely

translocated back to the perinuclear region and the cytoplasm with a concomitant decrease in the nuclei (fig. 8D).

At 2 min postinjury, the -isozyme is still localized to the

cytoplasm and the perinuclear region (fig. 8E) but by

10 min, a relocalization of the isozyme to the nuclei is

observed (fig. 8F) in some of the cells. By 30 min postinjury, the -isozyme is completely translocated to the nuclei384 Am J Nephrol 2003;23:380389 Polosukhina/Singaravelu/PadanilamControl 2 min 5 min 10 minFig. 6. PKC- expression in control and H2O2 treated cells during

and postinjury. LLCPK1 cells that were grown in DMEM only (control) is shown in A. B, C and D represent LLCPK1 cells that were

fixed at 2, 5 and 10 min, respectively, during the exposure to 500 M

H2O2. The images are shown at a magnification of !200. E, F and G

represent LLCPK1 cells that underwent 30 min of H2O2 mediated

injury followed by 2, 5 and 10 min of recovery in DMEM. PKC-

isozyme is localized to the cytoplasm and perinuclear region in control cells (A). At 2 min during the injury, the isozyme has a similar

localization as in control cells, although the staining in the perinuclear region is more pronounced. At 5 and 10 min during the injury,

the isozyme is translocated to the nuclei of the cells (C, D). The cells

from untreated, 2, 5 and 10 min during the injury were also stained

with the nuclear stain Hoechst 33258. High magnification (!1,200)

photographs of individual cells from each of these time points are

shown in E, F, G and H, respectively. At 2 minutes postinjury, PKC-

localizes to the nuclear membrane and perinuclear region (I) and its

localization is not altered at 5 (J) and 10 min (K) postinjury.(fig. 8G). In order to further confirm its translocation,

Western blot analysis was performed on proteins derived

from cytoplasmic and nuclear fractions of LLCPK1 cells

that underwent H2O2 injury. As shown in figure 9, PKC-

expression is observed both in the cytoplasmic and nuclear fractions at 2 min during the injury. The majority of

expression is observed in the nuclear fraction at 2 min. At

10 min during the injury, PKC- expression is limited to

the cytoplasmic fraction concurring with the immunocytochemistry data shown in figure 8D. At 10 min postinjury, translocation of a fraction of PKC- to the nucleus is

confirmed by its presence in both cytoplasmic and nuclear fractions. At 30 min postinjury, the complete translocation to the nucleus is further established by the presence of the majority of expression in the nuclear fraction.At 1 h postinjury almost all the cells have the PKC-

isozyme localized to the nuclear membrane and perinuclear region. At 2 and 3 h postinjury also, the PKC- isozyme is localized to the same region (fig. 10). Immunolocalization of the PKC- isozyme at 1, 2 and 3 h postinjuryPKC in Renal Oxidant Injury Am J Nephrol 2003;23:380389 385Fig. 7. Translocation of PKC- expression inH2O2 treated cells during and after injury wasconfirmed by Western blotting. At 5 min duringthe injury, a band representing PKC- is observed both in the cytoplasmic and nuclear fraction and its expression in the nuclear fraction isenhanced at 10 min during the injury. At 2 and10 min postinjury, the majority of PKC- expression is seen in the cytoplasmic fraction andalmost no expression of PKC- is observed inthe nuclear fraction. C = Cytoplasmic fraction;N = nuclear fraction.ControlDuring injuryPost injuryC

2 min

N C

10 min

NC

10 min

N C

30 min

Na2 min 5 min 10 min2 min 10 min 30 minFig. 8. PKC- expression in control and H2O2 treated cells during

and postinjury. PKC- expression in LLCPK1 cells that were grown

in DMEM only (control) is shown in A. B, C and D represent

LLCPK1 cells that were fixed at 2, 5 and 10 min, respectively, during

the exposure to H2O2. E, F and G represent LLCPK1 cells that underwent 30 min of H2O2 mediated injury followed by 2, 5 and 10 min of

recovery in DMEM. PKC- isozyme is localized to the cytoplasm,

perinuclear region and weakly along the plasma membrane in control

cells (A). At 2 min during the injury, the isozyme translocated to the

nuclear membrane and into the nucleus (B). At 5 min during the

injury, the isozyme is translocated to the nuclei of a large number of

cells (C). At 10 min during the injury, PKC- completely translocated

back to the perinuclear region and cytoplasm (D). At 2 min postinjury, PKC- localizes to the nuclear membrane and perinuclear region

(E). At 10 min, PKC- localizes to the nuclear membrane and to the

nuclei (F). At 30 min during the recovery period, PKC- translocated

back to the nuclei (G). Degree of magnification in all figures is at

!200.shows that the -isozyme is also localized to the nuclear

membrane and perinuclear region at these time points

(fig. 10).In order to determine if the protective effect of PKC

activators is mediated by an alteration in the translocation of PKC isozymes during or postinjury, the cells were

injured in presence of PMA or OAG. Inclusion of the activators in the media during the injury did not affect the

subcellular distribution of PKC- or at any time during

or postinjury. However, pretreatment of the cells with the

activators resulted in translocation of PKC- isozyme to

all regions of the plasma membrane (data not shown).386 Am J Nephrol 2003;23:380389 Polosukhina/Singaravelu/PadanilamFig. 9. Translocation of PKC- expression inH2O2 treated cells during and postinjury was

confirmed by Western blotting. At 2 min during

injury, majority of PKC- expression is in the

nuclear fraction while at 10 min, it is mostly in

the cytoplasmic fraction. At 10 and 30 min

postinjury, the expression of PKC- is mostly

observed in the nuclear fractions as shown by a

corresponding band. C = Cytoplasmic fraction;

N = nuclear fraction.During injuryPost injuryC

2 min

N C

10 min

NC

10 min

N C

30 min

Nz1 h 2 h 3 hazFig. 10. Expression of PKC- and PKC- at 1, 2 and 3 h postinjury. PKC- is localized to the nuclear membrane and perinuclear

region at 1 hour postinjury and its expression pattern is not altered at 2 and 3 h postinjury. PKC- has translocated from the nucleus

back to the nuclear membrane and perinuclear region at 1 h postinjury and is located in the same area at 2- and 3-hour time points.

Degree of magnification is !200 in all figures except for PKC- at 1 h, which is shown at a magnification of !400.DiscussionThe H2O2 injury model has been widely used as an

ischemic injury model by various investigators based on

the role of superoxides in mediating the injury. Renal epithelial cells when exposed to H2O2 are shown to induce

cell death by both apoptosis and necrosis [13, 14]. PKC is

activated by oxidative stress and antioxidants can inhibit

its function [15, 16]. The role of PKC isozymes was investigated by studying the effect of pharmacological activation or inhibition of the isozymes on the severity of the

injury using this in vitro model. The results presented inPKC in Renal Oxidant Injury Am J Nephrol 2003;23:380389 387this study show that LLCPK1 cells exposed to H2O2 are

less susceptible to oxidant injury upon activation of PKC

isozymes.The activation of PKC isozymes was achieved using

PMA, OAG, DOG or bryostatin to determine if PKC-

mediated signal transduction pathways may be involved

in hydrogen peroxide mediated oxidant injury. We have

surveyed the expression and activation pattern of PKC-,

I, II, and postinjury in LLCPK1 cells. The expression of PKC- I, II or is not altered showing that only

PKC- of the classic and PKC- of the atypical PKC family are activated by the injury. Phorbol esters and DAG

bind to the zinc finger structures in the C1 domain of

PKC molecules and activate their function possibly

through a conformational change. In classical and novel

PKCs the binding motif is duplicated as a direct repeat

contiguous sequence in their C1 domain while the atypical PKCs have only one zinc finger motif. Although it has

been shown that one of the zinc finger units is sufficient to

furnish phorbol ester binding, the presence of the single

binding motif in PKC- is insufficient to confer binding[17] suggesting that PKC- activation by the pharmacological agents used in this study may not play a role in

mediating the protective effect after H2O2 injury. This

suggests that PKC- activation by the phorbol esters may

confer the protective effect in this setting. However, it

should be noted that our data do not rule out the possibility of PMA or diacyl-glycerol activation of members of the

novel family of PKC and their participation in the pathways mediating H2O2 injury. In addition, the possibility

that these agents could activate another signaling pathway

leading to the release of arachidonic acid [18] or activation of the phosphatidylinositol 3-kinase (PI3-K) pathway[19] to activate PKC- cannot be ruled out.

To examine the effect of PKC inhibition on H2O2

mediated oxidant injury, we used the pan-specific PKC

inhibitors calphostin C or GF 109203X. Inhibition of the

isozymes using these specific inhibitors or by prolonged

exposure to PMA did not alter the susceptibility of the

cells to oxidant injury. It is unclear why the inhibition of

PKC isozymes did not exacerbate the injury. A likely

explanation is that PKC activation is not the exclusive

mechanism to protect the cells from injury. On the other

hand, in presence of activators, the overexpressed PKC

isozymes will be further activated and offers protection by

modulating the activity of their substrates. Thus the data

presented here demonstrates a central role for PKC activation in H2O2 mediated oxidant stress in LLCPK1 cells.The expression pattern of PKC- and - in the in vivo

model coincided with the expression profile in the in vitro

model used in this study [9]. In the rat model, expression

of both PKC- and - were highly induced in the first 1 h

after ischemic injury and its expression was down-regulated at later time points. A second phase of up-regulation

of PKC- and - expression was observed in the in vivo

model at 1 day postinjury and its higher level of expression was maintained at 5 and 7 days postinjury. LLCPK1

cells that underwent H2O2 injury showed that PKC-

expression was induced within the first 10 min of injury

(fig. 4, 5) and its expression was down-regulated to that of

normal levels by 15 to 30 min. A second phase of up-regulation of PKC- isozyme was observed at 2 hour postinjury and the higher level of expression was also observed at

24 hours postinjury. The similarity in expression pattern

of PKC- between the rat model and the LLCPK1 model

suggests that the isozyme may play a similar role in regulating the physiological functions in both models of the

injury. The expression pattern of PKC- in the rat model

also coincided with the in vitro model in the early phase of

the injury. The expression of PKC- was highly induced

within the first 15 min of injury (fig. 4, 5) in the in vitro

model and was down-regulated at 30 min and later time

points. However, unlike the in vivo model, a second phase

of up-regulation of PKC- was not observed in the in vitro

model.H2O2 injury triggered the translocation of PKC- and

PKC- from the cytoplasmic compartment to the nucleus

and the shuttling between the two locations at various

time points during and postinjury. The translocation of

PKC did not result from its activation by PMA or other

activating agents used in this study. We have previously

shown that in renal PTC, the PKC- isozyme is associated

with the apical/brush border membrane in sham operated

kidneys but when subjected to ischemic injury, the isozymes is found to translocate to both the plasma membrane and to the nucleus. Similarly PKC-II translocated

to the plasma membrane and PKC- to the apical membrane. The mechanisms that may mediate the translocation of PKC to various compartments are not addressed

in this study.Thus, the data presented here suggest that the differential expression and translocation of PKC- and PKC-

are involved in the H2O2 induced injury of LLCPK1 cells.

The signaling pathways by which PKC mediate the protective or deleterious effects in ischemic kidney or in in

vitro cell culture models are not elucidated. It has been

reported that mitogen-activated protein kinase (MAPK)

activation play a role in IRI in the kidney and IPC results

in much less activation of the MAPKs and stress activated

protein kinases [2, 20]. A role for PKC- in inducing388 Am J Nephrol 2003;23:380389 Polosukhina/Singaravelu/PadanilamMAPK activation through MEK in cardiomyocytes has

been reported [21]. The role of PKC in mediating the activation of MAPK in renal ischemia has not been investigated.PKC has been implicated in regulating various signal

transduction pathways leading to physiological alterations in renal tissue. Several lines of evidence suggest that

many of the renal tubular transporting systems are under

the control of PKC. Na+K+ATPase, which is of vital

importance for the reabsorption of sodium in all tubular

segments, is phosphorylated and inactivated by PKC in

vitro [22]. PKC stimulates the activity of the Na+-H+

exchanger and the Na-HCO3 co-transporter in renal cells[23].The specific role of PKC- and PKC- in mediating

cellular injury, protection or regeneration can only be

understood by studying the signaling pathways mediated

by them. Identification of downstream targets of PKC in

LLCPK1 cells upon injury by H2O2 will provide more

insight into the pathways triggered by the activation of the

isozymes. A better understanding of the physiological

substrates and the signal transduction pathways mediated

by individual PKC isozymes in ischemic renal injury is

necessary to modulate their functions for a better outcome in the setting of acute renal failure.AcknowledgementsThis work was supported by the NIH grant DK 52907 to B.J.P. A

portion of this work was presented at the annual meeting of the

American Society of Nephrology in San Francisco, Calif., USA and

has been published in abstract form [J Am Soc Nephrol

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