PROTEIN KINASE C MEDIATES REPAIR OF MITOCHONDRIAL AND TRANSPORT FUNCTIONS FOLLOWING TOXICANT-INDUCED INJURY IN RENAL CELLS.

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1 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet JPET Fst This Forwrd. rticle Pulished hs not een on Mrch copyedited 28, 2003 nd formtted. s DOI: /jpet The finl version my differ from this version. PROTEIN KINASE C MEDIATES REPAIR OF MITOCHONDRIAL AND TRANSPORT FUNCTIONS FOLLOWING TOXICANT-INDUCED INJURY IN RENAL CELLS. Grżyn Nowk University of Arknss for Medicl Sciences Deprtment of Phrmceuticl Sciences 4301 West Mrkhm Street Little Rock, AR Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 Copyright 2003 y the Americn Society for Phrmcology nd Experimentl Therpeutics.

2 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Running title: Role of PKC in the repir of renl cell functions Address for correspondence: Grżyn Nowk, Ph.D University of Arknss for Medicl Sciences Deprtment of Phrmceuticl Sciences 4301 West Mrkhm St., MS Little Rock, AR Phone: ; Fx: E-mil: gnowk@ums.edu Mnuscript Informtion: Text Pges = 30 Numer of Tles = 0 Numer of Figures = 8 Numer of References = 40 Numer of Words in Astrct = 227 Numer of Words in Introduction = 747 Numer of Words in Discussion = 1498 Arevitions: RPTC, renl proximl tuulr cells; TBHP, tert-utylhydroperoxide; DCVC, S-(1,2- dichlorovinyl)-l-cysteine; ARF, cute renl filure; PKC, protein kinse C, PMA, phorol-12- myristte-13-cette; DMEM:F12, Dulecco's Modified Egle's Essentil medium (DMEM) : Hm's F-12 nutrient mix; Hepes, N-2-hydroxyethylpiperzine-N -2-ethnesulfonic cid; PMSF, phenylmethylsulfonyl fluoride; MBP, myelin sic protein; EDTA, ethylenediminetetrcetic cid; EGTA, ethylene glycol-is-(β-minoethylether)-n,n,n,n -tetrcetic cid; TBS, Trisuffered sline; QO 2, oxygen consumption; FCCP, p-trifluoromethoxyphenyl-hydrzone; MGP, α- D-glucopyrnoside. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 Recommended Section Assignment : Toxicology 2

3 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. ABSTRACT Previously, we hve shown tht renl proximl tuulr cells (RPTC) recover physiologicl functions following injury induced y the oxidnt, tert-utylhydroperoxide (TBHP), ut not y the nephrotoxic cysteine conjugte dichlorovinyl-l-cysteine (DCVC). This study exmined the role of protein kinse C (PKC) in the repir of RPTC functions following sulethl injury produced y these toxicnts. Totl PKC ctivity decresed 65% nd 86% following TBHP nd DCVC exposures, respectively, nd recovered in TBHP-injured ut not in DCVC-injured RPTC. Mitochondril function, ctive N + trnsport, nd N + -dependent glucose uptke decresed following toxicnt exposure nd recovered in TBHP- ut not in DCVC-injured RPTC. PKC inhiition decresed the repir of RPTC functions following TBHP injury. PKC ctivtion promoted recovery of mitochondril function nd ctive N + trnsport in TBHPnd DCVC-injured RPTC ut hd no effect on recovery of N + -dependent glucose uptke. We conclude tht in RPTC: 1) totl PKC ctivity decreses following TBHP nd DCVC injury nd recovers following TBHP ut not fter DCVC exposure, 2) recovery of PKC ctivity precedes the return of physiologicl functions fter oxidnt injury, 3) PKC inhiition decreses recovery of physiologicl functions, nd 4) PKC ctivtion promotes recovery of mitochondril function nd ctive N + trnsport ut not N + - dependent glucose uptke. These results suggest tht the repir of renl functions is medited through PKC-dependent mechnisms nd tht cysteine conjugtes my inhiit renl repir, in prt, through inhiition of PKC signling. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

4 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Numerous toxicnts cn cuse renl dysfunction through their ility to induce sulethl injury to renl proximl tuulr cells (RPTC), which results in decresed norml cellulr functions without producing cell deth nd loss. Mitochondril dysfunction followed y ATP depletion, reduced metolic functions, nd decreses in N + -K + -ATPse ctivity nd ctive N + trnsport re the mjor ltertions in RPTC injured y different toxicnts (Elfr et l., 1986; Lsh nd Anders, 1987; Schnellmnn, 1988, Lsh et l., 1995; Nowk et l., 1998; Nowk et l., 1999; Lsh et l., 2001). Oxidtive stress hs een implicted in the mechnism of the renl dysfunction ssocited with ischemi/reperfusion nd the nephrotoxicity of numer of drugs nd environmentl chemicls including hlocrons. Tert-utylhydroperoxide (TBHP) hs een commonly used s model oxidnt in in vitro models of RPTC (Schnellmnn, 1988; Kys nd Schnellmnn, 1995, Nowk nd Schnellmnn, 1997; Blig et l., 1997, Nowk et l., 1998). Exposure of RPTC to TBHP results in the formtion of rective oxygen species, lipid peroxidtion, mitochondril dysfunction, nd impired trnsport functions (Borkn et l. 1989; Schnellmnn, 1988; Nowk et l., 1998). Hlogented hydrocrons represent lrge group of environmentl chemicls tht re used s chemicl intermedites, solvents, nd pesticides nd produce toxicity nd cute renl filure (ARF) fter their enzymtic conversion to rective intermedites nd the formtion of nephrotoxic cysteine S-conjugtes (Elfrr et l., 1986; Lsh nd Anders, 1987). Dichlorovinyl-L-cysteine (DCVC) is model hlocron nephrotoxicnt tht is selective for RPTC nd produces injury through multiple mechnisms including mitochondril dysfunction (Lsh nd Anders, Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, ; Stevens et l., 1986; Vmvks et l., 1992; vn der Wter et l., 1994; Chen et l., 2001). The kidney hs the potentil for complete recovery from ARF (Tock, 1992). Using n in vitro model of primry cultures of RPTC, we hve shown tht RPTC proliferte nd recover physiologicl functions following sulethl injury induced y the oxidnt TBHP (Nowk et l., 1998). However, the repir of RPTC functions does not occur in sulethlly injured RPTC fter DCVC exposure (Nowk et l., 1999). DCVC lso decreses synthesis of extrcellulr mtrix proteins of the RPTC sement 4

5 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. memrne, including collgen IV, nd disrupts locliztion of collgen inding integrins (Nowk et l., 2000; Nony et l. 2001; Nony nd Schnellmnn, 2002). Interestingly, EGF nd phrmcologicl concentrtions of scoric cid promote the repir of mitochondril function nd ctive N + trnsport in DCVC-injured RPTC (Nowk et l., 1999; 2000; Nony et l., 2001). The promotion of RPTC repir y scoric cid is medited, in prt, y collgen IV nd is ssocited with the re-locliztion of collgen-inding integrins to the sement memrne (Nony et l., 2001). Protein kinse C (PKC), fmily of serine/threonine protein kinses, controls 70% of the phosphorylting ctivity in renl proximl tuules (Koryn et l., 1994) nd regultes numerous physiologicl functions of renl epithelil cells including gluconeogenesis, N + /K + -ATPse ctivity, nd the trnsport of mino cids, glucose, sodium, potssium, chloride, phosphte, wter, nd orgnic nions nd ctions (Dempsey et l., 2000). Recent studies suggest tht PKC is lso involved in the regultion of cell survivl nd drug-induced cell injury (Dempsey et l., 2000). PKC is trget for some toxicnts. Short-time exposure to oxidnts ctivtes PKC in some cell types (O Brin et l., 1988; Plumo et l., 1992; Ae et l., 1998). In contrst, longer exposures to oxidnts inctivte PKC y modifying its ctlytic domin (Goplkrishn nd Anderson, 1987). PKC signling regultes the regenertive processes in the liver following prtil heptectomy (Dller et l., 1994; Tessitore et l., 1995). PKC lso hs een implicted in the renl recovery following ARF (Alerti et l., 1993; LPort nd Commoli, 1993) nd in wound heling following Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 mechniclly-induced injury in renl tuulr epithelil cells (Sponsel et l., 1995). Renl regenertion following toxicnt exposure is ssocited with differentil modultion of PKC isozymes. Activtion of PKCδ, PKCε nd PKCζ, ut not PKCα or PKCβ occurs during compenstory renl hypertrophy induced y unilterl nephrectomy (Dong et l., 1993). Regenertion following folic cid- nd DCVCinduced injury in the kidney is ssocited with down-regultion of PKCα nd PKCζ (Dong et l., 1993; Zhng et l., 1993). Although PKC ppers to e involved in renl recovery following toxic 5

6 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. insult, it is not known wht functions nd processes re regulted y this kinse in RPTC. Therefore, the im of this study ws to determine whether PKC plys role in the recovery of mitochondril function, ctive N + trnsport, nd N + -coupled glucose uptke in RPTC following sulethl injury induced y two different toxicnt, the oxidnt, TBHP, nd the model hlocron, DCVC. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

7 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. MATERIALS AND METHODS Animls. Femle New Zelnd White rits ( kg) purchsed from Myrtle's Ritry (Thompson Sttion, TN) were used in this study. Chemicls nd regents. S-(1,2-dichlorovinyl)-L-cysteine (DCVC) ws synthesized ccording to the method of Moore nd Green (1988). L-Ascoric cid-2-phosphte mgnesium slt ws purchsed from Wko BioProducts (Richmond, VA). Protein kinse C ssy kit, phorol-12-myristte-13-cette (PMA), nd cell culture medi were otined from Gico Invitrogen Corportion (Grnd Islnd, NY). Cell culture hormones, tert-utyl hydroperoxide, nd protese nd phosphtse inhiitors were otined from Sigm (St. Louis, MO). Clphostin C ws purchsed from Cliochem (L Joll, CA). Cell permele PKC peptide inhiitor, myristoylted protein kinse C frgment (20-28) (myr-phe-al-arg-lys-gly-al-leu- Arg-Gln; pseudosustrte sequence from PKC) ws supplied y BIOMOL Reserch Lortories (Plymouth Meeting, PA). [γ- 32 P]ATP (S.A Ci/mmol) nd methyl α-d-glucopyrnoside, [glucose- 14 C(U)] (S.A. 282 mci/mmol) were purchsed from Amershm Biosciences (Pisctwy, NJ) nd DuPont New Englnd Nucler (Boston, MA), respectively. PKC-α, PKC-δ, PKC-ε, nd PKC-ζ ntiodies were otined from BD Trnsduction Lortories (Sn Diego, CA). Antiodies ginst phosphorylted forms of PKC-α nd PKC-ε were supplied y Upstte (Lke Plcid, NY). Anti-mouse IgG coupled to horserdish peroxidse ws supplied y Kirkegrd & Perry Lortories (Githersurg, MD) nd Supersignl Chemiluminescent Sustrte y Pierce (Rockford, IL). The sources of the other regents Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 hve een descried previously (Nowk nd Schnellmnn, 1996). Isoltion of proximl tuules nd culture conditions. Rit renl proximl tuules were isolted y iron oxide perfusion method nd grown in 35 mm culture dishes in improved conditions s descried previously (Nowk nd Schnellmnn, 1996). The purity of the renl proximl tuulr S 1 nd S 2 segments isolted y this method is pproximtely 96%. The culture medium ws 50:50 mixture of Dulecco's modified Egle's essentil medium (DMEM) nd Hm's F-12 nutrient mix without phenol red, pyruvte, 7

8 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. nd glucose, supplemented with 15 mm NHCO 3, 15 mm N-2-hydroxyethylpiperzine-N -2- ethnesulfonic cid (Hepes), nd 6 mm lctte (ph 7.4, 295 mosmol/kg). Humn trnsferrin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (50 nm), ovine insulin (10 nm), nd L-scoric cid-2-phosphte (0.05 mm) were dded to the medium immeditely efore dily medi chnge (2 ml/dish). Toxicnt tretment of RPTC monolyer. RPTC cultures reched confluence within 6 dys nd were treted with toxicnts on the seventh dy of culture. RPTC were treted with 0.2 mm TBHP for minutes or with 0.2 mm DCVC for 1.5 hr to otin pproximtely 25% cell deth nd loss. Following toxicnt exposure, the remining cellulr monolyer ws wshed with fresh culture medium nd cultured for the following 4 dys. PKC inhiitors (100 nm clphostin C nd 20 µm myristoylted protein kinse C (20-28) peptide) were dded dily strting with the medi chnge following TBHP exposure. PKC ctivtor, 100 nm PMA, ws dded 30 min prior to toxicnt nd ws present in the medi for 24 hours following toxicnt exposure. Smples of RPTC were tken t vrious time points fter toxicnt exposure for mesurements of cellulr functions. Isoltion of cytosolic nd memrne frctions. RPTC smples were hrvested t vrious time points during recovery period following toxicnt exposure. Monolyers were wshed 4 times with ice-cold PBS (ph 7.4) to remove ll non-vile cells, cells scrped from the dishes, suspended in 1 ml PBS, nd pelleted y centrifugtion. RPTC pellet ws resuspended in ice-cold isoltion uffer (20 mm Tris-HCl, ph 7.5, contining 10 mm MgCl 2, 80 mm β-glycerophosphte, 2 mm EGTA, 2 mm EDTA, 2 mm dithiothreitol, Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, mm phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml protinin, 5 µg/ml pepsttin, nd 5 µg/ml leupeptin). The cells were riefly sonicted (3x5 sec) on ice nd centrifuged t 1,000 x g for 5 min to remove cell deris nd nuclei. The superntnt ws spun down t 100,000xg for 30 min t 4 o C nd the superntnt resulting from this centrifugtion represented cytosolic frction. The pellet ws resuspended in the isoltion uffer contining 1% Triton X-100, incuted on ice for 30 min, nd centrifuged t 100,000xg for 30 min t 4 o C. The superntnt resulting from this spin represented the prticulte frction. 8

9 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Mesurement of PKC ctivity. The ctivity of PKC ws sed on mesurement of the phosphoryltion of synthetic peptide derived from the myelin sic protein (MBP) sequence (4-14) s descried y Ysud et l. (1990). The N-terminl glutmine of this peptide hs een cetylted in order to mintin the peptide s stility (Ac-MBP(4-14)). Specificity of the phosphoryltion of Ac- MBP(4-14) y PKC ws confirmed y using the PKC pseudosustrte inhiitor peptide PKC(19-36), which cts s potent inhiitor for this sustrte. PKC ctivity ws mesured in the rection mixture tht contined 20 mm Tris (ph 7.5), 20 mm MgCl 2, 1.0 mm CCl 2, 0.28 mg/ml phosphtidyl-l-serine, 10 µm PMA, 1 mm dithiotreitol, pepsttin, leupeptin, protinin (25 µg/ml ech), 1 mm PMSF, 40 mm β-glycerophosphte, 0.3% Triton X-100, 50 µm Ac-MBP(4-14), 20 µm [γ- 32 P]ATP, nd smple. Specificity of the phosphoryltion of Ac-MBP(4-14) y PKC ws mesured in the rection mixture contining ll ove components nd 20 µm PKC(19-36) peptide inhiitor. The rection ws crried out for 5 min t 30 o C nd terminted y spotting n liquot of rection mixture onto P-81 phosphocellulose discs. Following wshing the discs, γ- 32 P incorportion into sustrte ws determined y liquid scintilltion spectrometry. Specific PKC ctivity ws clculted s the PKC(19-36) inhiitorsensitive ctivity. Non-specific ctivity (ckground) ws determined in smples incuted in the sence of ctivtors (phosphtidylserine, PMA, CCl 2 ). Immunolotting - Immunolot nlysis ws used for the mesurement of protein levels of PKC α, PKC δ, PKC ε, nd PKC ζ in the cytosolic nd prticulte frctions of RPTC nd for ssessment of Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 levels of phosphorylted forms of PKC-α nd PKC-ε in totl RPTC homogentes. Smples of cytosolic nd prticulte frctions nd cell homogentes were lysed nd oiled for 10 min in Lemmli smple uffer (60 mm Tris-HCl, ph 6.8 contining 2% SDS, 10% glycerol, 100 mm β-mercptoethnol, nd 0.01 % romophenol lue) (Lemmli, 1970). Proteins were seprted using SDS-PAGE. Following electrolotting of the proteins to nitrocellulose memrne, lots were locked for 1 hr in Tris-uffered sline (TBS) uffer contining 0.5% csein nd 0.1% Tween 20 (locking uffer), nd incuted 9

10 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. overnight t 4 o C in the presence of primry ntiodies diluted in the locking uffer. Following wshing with TBS contining 0.05% Tween-20, the memrnes were incuted for 1 hr with nti-rit or ntimouse IgG coupled to horserdish peroxidse nd wshed gin. The supersignl chemiluminescent system ws used for protein detection. The results were quntified using scnning densitometry. Oxygen consumption. RPTC monolyers were wshed with 37 o C culture medium nd gently detched from the dishes with ruer policemn, suspended in wrm (37 o C) culture medium nd trnsferred to the oxygen consumption (QO 2 ) mesurement chmer. QO 2 ws mesured polrogrphiclly in RPTC suspended in the culture medium using Clrk type electrode s descried previously (Nowk nd Schnellmnn, 1996; Nowk et l., 1998; 1999; 2000). Bsl QO 2 ws used s mrker of entire mitochondril function. Oligomycin-sensitive QO 2 ws used s mker of oxidtive phosphoryltion. Oligomycin-sensitive QO 2 ws determined following ddition of oligomycin (1 µg/ml) nd ws clculted s the difference etween sl nd oligomycin-insensitive QO2. Uncoupled QO 2 ws used s mrker of integrity of the electron trnsport chin nd ws mesured following the ddition of cronyl cynide p-trifluoromethoxyphenyl-hydrzone (FCCP, 1.5 µm). Active N + trnsport. Active N + trnsport ws mesured using the ouin-sensitive QO 2 s mrker s descried previously (Nowk nd Schnellmnn, 1996; Nowk et l., 1998; 1999; 2000). Ouin-sensitive QO 2 ws mesured s the difference etween sl QO 2 nd QO 2 in the presence of Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, mm ouin. N + -coupled glucose uptke. N + -coupled glucose uptke ws ssessed using the non-metolizle glucose nlogue methyl α-d-glucopyrnoside (MGP) s descried previously (Nowk nd Schnellmnn, 1996). MGP uptke ws mesured in glucose-free medium used for RPTC culture nd corrected for N + -independent (phlorizin-insensitive) nd zero time uptkes. Protein ssy. Protein concentrtion in ll smples ws determined using icinchoninic cid ssy (BCA) with ovine serum lumin s the stndrd. 10

11 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Sttisticl nlysis. Dt re presented s mens ± SE nd were nlyzed for significnce using ANOVA. Multiple mens were compred using Student-Newmn-Keuls test. Sttements of significnce were sed on P<0.05. Renl proximl tuules isolted from n individul rit represented seprte experiment (n = 1) consisting of dt otined from 2 pltes. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

12 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. RESULTS The effect of TBHP nd DCVC exposures on PKC ctivity in the cytosolic nd prticulte frctions of RPTC. Totl PKC ctivity decresed y 60% nd 63% in the cytosolic nd prticulte frctions of sulethlly-injured RPTC, respectively, during the 24 hr following TBHP exposure (Fig. 1A nd B). PKC ctivity in the cytosol returned to control levels on dy 2 of the recovery period (Fig. 1A). However, PKC ctivity in the prticulte frction of RPTC did not recover until dy 4 following TBHP-induced injury (Fig. 1B). DCVC exposure decresed PKC ctivity in oth the cytosolic nd prticulte frctions of RPTC ut the DCVC-induced decreses in PKC ctivity were slower tht those induced y TBHP. At 24 hr following the DCVC tretment, PKC ctivity ws decresed y 86% in oth frctions (Fig. 2 A nd B). In contrst to TBHP-induced injury, PKC ctivity in RPTC did not recover fter DCVC-injury (Fig. 2 nd 6). PKC nd the recovery of mitochondril function. Bsl, uncoupled, nd oligomycin-sensitive QO 2 s were used s mrkers of mitochondril function in RPTC. Specificlly, uncoupled QO 2 served s mrker of the electron trnsport rte wheres oligomycin-sensitive QO 2 ws used s mrker of the oxidtive phosphoryltion. TBHP tretment decresed sl, uncoupled, nd oligomycin-sensitive QO 2 s y 41%, 52%, nd 48%, respectively, t 4 hr following the exposure (Fig. 3 nd 4). Respirtory functions of RPTC returned to control levels on dy 4 of the recovery period (Fig. 3 nd 4). The return Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 of mitochondril function followed the recovery of PKC ctivity in the cytosol nd ws ccompnied y the recovery of PKC ctivity in the prticulte frction (Fig. 1, 3, nd 4). To determine whether PKC plys role in the recovery of mitochondril function following TBHP-induced injury RPTC were treted with specific PKC inhiitor, clphostin C (100 nm), nd the return of sl QO 2, exmined during the recovery period. Clphostin C hd no effect on sl QO 2 in control RPTC or on the decrese in QO 2 in TBHP-treted RPTC, ut it inhiited the recovery of this 12

13 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. function in sulethlly-injured RPTC (Fig. 3). Likewise, the tretment of RPTC with nother specific PKC inhiitor (PKC (20-28)), peptide derived from pseudosustrte sequence of PKC, prevented the recovery of sl QO 2 (Fig. 4A). Furthermore, PKC (20-28) inhiited the return of oligomycinsensitive nd uncoupled QO 2 s in TBHP-injured RPTC (Fig. 4 B nd C). Experiments were lso performed to determine whether ctivtion of PKC ccelertes the repir of mitochondril function following TBHP injury. PMA (100 nm) ws used to ctivte PKC in RPTC prior to toxicnt-induced injury. Protein levels of PKCα nd PKCε in the prticulte frction of RPTC incresed 1.3-fold nd 2.5-fold, respectively, within 2 to 5 minutes of PMA tretment (Fig. 5A). Protein levels of PKCα nd PKCε in the cytosolic frction of PMA-treted RPTC decresed concomitntly (Fig. 5A). The levels of phosphorylted forms of PKCα nd PKCε were incresed 2.5 nd 4.4-fold, respectively, in RPTC treted for 5 minutes with 100 nm PMA wheres the totl levels of PKCα nd PKCε remined unffected (Fig. 5B). These dt show tht PMA induces phosphoryltion nd trnsloction of PKCα nd PKCε from the cytosol to the prticulte frction, which suggests tht PMA ctivtes these PKC isozymes. No phosphoryltion nd trnsloction of PKCδ nd PKCζ occurred in the presence of PMA in RPTC (dt not shown). PMA tretment prior to TBHP exposure did not protect ginst the decrese in mitochondril function following the exposure (Fig. 5B). However, the recovery of mitochondril function in PMA-treted RPTC occurred on dy 2 following Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 TBHP injury nd ws ccelerted in comprison with the recovery of this function in RPTC pretreted with the vehicle (dy 4) (Figs. 5B nd 4A). DCVC exposure decresed sl QO 2 in sulethlly injured RPTC y 33% nd 47% t 4 nd 24 hr, respectively. Oligomycin-sensitive QO 2 decresed y 77% (21.7 ± 1.1 vs. 5.1 ± 3.2 nmol O 2 /min/mg protein in control nd DCVC-injured RPTC, respectively) nd uncoupled QO 2 decresed 63% (82.3 ± 5.6 vs ± 3.7 nmol O 2 /min/mg protein in control nd DCVC-injured RPTC, respectively) t 4 hr 13

14 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. following the exposure. No repir of sl QO 2 occurred following DCVC exposure (Fig. 5C). However, tretment with PMA prior to DCVC exposure resulted in the return of sl QO 2 within 2 dys in injured RPTC (Fig. 5C). These results demonstrte tht inhiition of PKC prevents the repir of mitochondril function following toxicnt injury in RPTC nd tht ctivtion of PKC prior to toxicnt exposure promotes recovery of respirtory functions. Thus, these dt suggest tht PKC plys role in the repir of mitochondril function following toxic insult in RPTC. PKC nd the recovery of ctive N + trnsport. Active N + trnsport ws used s mrker of the solterl memrne function nd ws ssessed y mesurement of ouin-sensitive QO 2. Active N + trnsport is n ATP-consuming process s it is driven y N + /K + -ATPse. The consumption of oxygen ssocited with production of ATP required for mintining the N + /K + -ATPse ctivity nd ctive N + trnsport ccounts for pproximtely 50% of sl oxygen consumption in RPTC. Ouin, specific N + /K + -ATPse inhiitor, decreses oxygen consumption y the mount of oxygen ssocited with the synthesis of ATP tht is consumed y N + /K + -ATPse nd ctive N + trnsport. Therefore, ouin-sensitive portion of QO 2 is n indirect indictor of ctive N + trnsport. Ouin-sensitive QO 2 decresed (51%) t 4 hr following TBHP exposure nd returned on dy 4 of the recovery period (Fig. 6A). Inhiition of PKC (using clphostin C or PKC (20-28)) did not ffect decreses in ouin-sensitive QO 2 t 4 hr following TBHP injury ut prevented the return of this Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 function on dy 4 of the recovery period (Figs. 6A nd 6B). In contrst, the return of ouin-sensitive QO 2 in RPTC treted with PMA prior to TBHP exposure occurred on dy 2 nd ws ccelerted in comprison with RPTC exposed to TBHP only (Fig. 7A). DCVC exposure in RPTC resulted in 40% decrese in ouin-sensitive QO 2 t 4 hr of the recovery period (Fig. 7B). Ouin-sensitive QO 2 did not recover over time (Fig. 7B). However, 14

15 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. tretment with PMA prior to DCVC exposure resulted in the return of ouin-sensitive QO 2 in RPTC within 2 dys following the exposure (Fig. 7B). These dt show tht PKC inhiition prevents the recovery of ctive N + trnsport following oxidnt injury in RPTC nd tht PKC ctivtion promotes the repir of ctive N + trnsport following toxicnt exposure. Interestingly, inhiition of PKC y PKC (20-28) peptide decresed ouin-sensitive QO 2 t 4 hr, wheres clphostin C hd no effect on this function in control RPTC (Fig. 6). This could e explined y dissimilr sensitivity of different PKC isozymes to these two inhiitors. PKC (20-28) is more specific nd generl PKC inhiitor wheres clphostin C is thought to inhiit primrily novel (C 2+ - independent nd PMA-sensitive) isozymes of PKC. PKC nd the recovery of N + -dependent glucose uptke. N + -dependent glucose uptke ws used s mrker of the rush-order memrne function in RPTC. TBHP nd DCVC decresed N + - dependent glucose uptke y 66% nd 63%, respectively, t 4 hr following the exposure (Fig. 8A nd 8B). N + -dependent glucose uptke recovered following TBHP- ut not following DCVC-induced injury (Fig. 8A nd 8B). Inhiition of PKC with clphostin C prior to TBHP exposure inhiited the return of N + -dependent glucose uptke in regenerting RPTC (Fig. 8A). Furthermore, 4-dy tretment of RPTC with clphostin C inhiited N + -dependent glucose uptke y 89% (Fig. 8A). Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 Activtion of PKC using PMA did not ccelerte the repir of N + -dependent glucose uptke in TBHP-injured RPTC (Fig. 8B). Likewise, tretment of RPTC with PMA prior to DCVC injury did not promote the recovery of N + -dependent glucose uptke (Fig. 8B). 15

16 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. DISCUSSION Toxicnt-induced renl dysfunction is often cused y chemiclly produced sulethl injury to RPTC, which decreses the physiologicl functions of renl proximl tuules without producing pprent cell deth. It hs een lso proposed tht some toxicnts cuse renl dysfunction not only y inducing injury ut lso y inhiiting RPTC regenertion (Counts et l., 1995). Our previous study showed tht RPTC in primry culture recover their functions following sulethl injury induced y n oxidnt (TBHP) nd tht this repir is medited y utocrine mechnisms (Nowk et l., 1998). In contrst, physiologicl functions do not recover in RPTC following injury produced y the hlocron nephrotoxicnt DCVC (Nowk et l., 1999). This suggested tht DCVC disrupts the utocrine mechnisms of RPTC repir. The repir of mitochondril function nd ctive N + trnsport ut not N + -dependent glucose trnsport in DCVC-injured RPTC re promoted y EGF, phrmcologicl concentrtions of scoric cid, nd collgen IV (Nowk et l., 1999; Nowk et l., 2000; Nony et l., 2001). Therefore, our previous dt suggested tht different toxicnts ffect the mechnisms of RPTC repir in differentil mnner. The present study exmined the role of one of the signling molecules, PKC, in the recovery of RPTC functions following toxicnt-induced injury. The trnsmission of intrcellulr signls is medited y network of intercting proteins tht regulte vriety of cellulr processes nd functions. PKC, fmily of serine/threonine protein kinses, is criticl element of this network (Dempsey et l., 2000). Toxicnts hve een implicted in Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 the ltertions in PKC ctivity. Our results show tht TBHP injury in RPTC is ssocited with decreses in totl PKC ctivity. These dt remin in contrst to erly nd trnsient ctivtion of PKC in neuronl nd endothelil cells in response to short-term exposure to n oxidnt (O Brin et l., 1988; Plumo et l., 1992; Ae et l., 1998). In contrst, longer exposure to oxidtive stress modifies the ctlytic domin of PKC nd inctivtes PKC (Goplkrishn nd Anderson, 1987), which is consistent with our results in RPTC. DCVC-induced decreses in totl PKC ctivity in our model re 16

17 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. in greement with the down-regultion of renl PKCα following n exposure to DCVC in vivo nd with the inhiition of PKC ctivity in the kidney y exposure to folic cid in vivo (Dong et l., 1993). PKC hs lso een implicted in regenertion of different tissues nd orgns. Liver regenertion following prtil heptectomy nd cron tetrchloride injury is ssocited with the ctivtion of PKCα, PKCβ, PKCδ, nd PKCζ (Dller et l., 1994; Tessitore et l., 1995; Sski et l., 1987). In the kidney, the ctivtions of PKCδ, PKCε, nd PKCζ, ut not PKCα or PKCβ occur during compenstory renl hypertrophy induced y unilterl nephrectomy (Dong et l., 1993). PKC ctivtion lso plys role in the ccelertion of wound heling following mechnicl injury in renl tuulr epithelil cells (Sponsel et l., 1995). In our RPTC model, decresed PKC ctivity following TBHP- nd DCVC-induced injury ws ccompnied y mjor decreses in the mitochondril function, ctive N + trnsport nd N + -dependent glucose uptke. The repir of RPTC functions following TBHP-induced injury ws preceded y the recovery of totl PKC ctivity. On the other hnd, the lck of repir of RPTC functions in DCVC-injured RPTC ws ssocited with the lck of return of the PKC ctivity. Furthermore, inhiition of PKC prevents the return of mitochondril function, ctive N + trnsport nd N + -dependent glucose uptke in TBHP-injured RPTC, which suggests tht PKC plys role in the repir of RPTC functions following the oxidnt injury. Two dissimilr PKC inhiitors hve een used in this study, the chemicl inhiitor clphostin C nd short peptide derived from PKCα nd PKCβ pseudosustrte sequence. PKC(20-28) inhiitor Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 decresed wheres clphostin C hd no effect on mitochondril function nd ctive N + trnsport in control RPTC, which could e explined y higher specificity of PKC(20-28) towrds PKCα nd PKCβ. The fct tht clphostin C, which is rther n inhiitor of novel PKC isozymes (δ, ε, η, µ, nd θ), did not hve ny effect on these functions in control RPTC suggests tht the mintennce of these functions is dependent on clssicl PKC isozymes. 17

18 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. The lck of functionl repir in RPTC treted with PKC inhiitors ws not due to toxic effects produced y these compounds. These inhiitors did not potentite decreses in mitochondril function nd ctive N + trnsport in TBHP-injured RPTC. Furthermore, oth inhiitors produced similr degree of inhiition of the repir of mitochondril function nd ctive N + trnsport. Interestingly, 4- dy tretment of control RPTC with clphostin C decresed N + dependent glucose uptke in RPTC y 89%, which suggests tht functionl PKC is necessry to mintin N + -dependent glucose trnsport in control RPTC. Becuse our dt suggested tht functionl PKC is criticl for the recovery of RPTC functions following toxic insult, we hypothesized tht ctivtion of PKC prior to toxicnt exposure my prevent the decreses in RPTC functions or promote the repir of some or ll RPTC functions. Activtion of PKC is ssocited with redistriution from the cytosol to cellulr memrnes nd orgnelles. This event is necessry for phosphoryltion of vrious intrcellulr proteins trgeted y PKC. Previous studies suggested tht renl regenertion following toxicnt exposure is ssocited with differentil modultion of PKC isozymes, which my e ssocited with different functions of these isozymes in the process of RPTC regenertion (Dong et l., 1993). At the present, however, the function of individul PKC isozymes in the repir of these functions is unknown. In our RPTC model, mong 4 mjor PKC isozymes present in RPTC (PKCα, PKCδ, PKCε nd PKCζ), only PKCα nd PKCε were phosphorylted nd redistriuted from the cytosol to the prticulte frction in response to Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 PMA tretment. In contrst, PMA hd no effect on the ctivtion of ERK1/2 (dt not shown). This suggested tht only PKCα nd PKCε were ctivted y PMA. PMA pretretment hd no effect on the decrese in mitochondril function induced y TBHP or DCVC, which demonstrtes tht PKC ctivtion does not protect ginst mitochondril dysfunction cused y these nephrotoxicnts. However, the recovery of mitochondril function ws ccelerted in TBHP-injured RPTC pretreted with PMA. Furthermore, PKC ctivtion olished inhiition of the repir of mitochondril function in 18

19 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. DCVC-injured RPTC. These results show tht PKC plys n importnt role in the repir of mitochondril function nd suggest tht PKCα nd/or PKCε re involved in these processes. Likewise, our dt show tht the repir of ctive N + trnsport following toxicnt injury is controlled y PKC. In contrst to the lck of repir of ctive N + trnsport in DCVC-injured RPTC, the recovery of this function occurred in DCVC-injured RPTC pretreted with PMA. Moreover, PKC ctivtion ccelerted the repir of ctive N + trnsport following TBHP injury, which ws not due to the protection ginst TBHP- nd DCVC-induced decreses in this function ecuse the degree of injury ws equivlent in RPTC treted with toxicnts nd RPTC treted with oth PMA nd toxicnts. Active N + -trnsport in RPTC is medited through N + /K + -ATPse, which is regulted through phosphoryltion y PKC nd PKA (Bertorello nd Aperi, 1989; Feschenko nd Swedner, 1994; Chilin et l., 1997). Phosphoryltion of N + /K + -ATPse leds to internliztion of the pump into endosomes nd decresed expression on the solterl memrne (Chilin et l., 1997). Our dt suggest tht this is lso true in our model s the short-term exposure to PMA (which results in PKC ctivtion) decresed ouin-sensitive QO 2 in control RPTC. In contrst, long-term exposure to PMA (24 hr), which does not ctivte PKC (Fig. 5A), hd no effect on ouin-sensitive QO 2. PKC ctivtion ppers to e necessry for promoting the recovery of ctive N + trnsport following toxicnt exposure. Previously, it hs een proposed tht the endosomes my constitute reservoirs Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 during N + /K + pump synthesis nd degrdtion (Chilin et l., 1997). It is likely tht N + /K + -ATPse cn e either protected or repired with higher efficiency in the endosomes nd is, therefore, ville for ressemly on the solterl memrne sooner during the recovery process. It is lso likely tht the ccelerted return of ctive N + trnsport y PKC ctivtion is due to the promotion of mitochondril function nd ATP vilility. N + /K + -ATPse ctivity requires constnt supply of ATP. The lck of recovery of ctive N + trnsport in DCVC-treted RPTC is ssocited with decresed ATP levels (Nowk et l., 1999). Promotion of the recovery of mitochondril function in DCVC-injured 19

20 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. RPTC treted with PMA my support the recovery of ctive N + trnsport y supplying ATP for N + /K + -ATPse function. In contrst, ctivtion of PKC did not promote the repir of N + -dependent glucose uptke in DCVC-injured RPTC. These results suggest tht the disruption of N + homeostsis following DCVC injury is not the exclusive mechnism responsile for the inhiition of repir of N + -dependent glucose uptke. Furthermore, our present dt suggest tht the recovery of this function lso involves PKCindependent mechnisms. In conclusion, our results show tht toxicnt-induced injury in RPTC cuses decreses in mitochondril function, ctive N + trnsport nd N + -dependent glucose uptke nd tht these events re ssocited with decreses in PKC ctivity. PKC inhiition locks wheres PKC ctivtion promotes the repir of mitochondril function nd ctive N + trnsport. In contrst, ctivtion of PKC does not stimulte the recovery of N + -dependent glucose uptke. Thus, our dt show tht PKC medites the repir of mitochondril function nd ctive N + trnsport following toxicnt injury nd suggest tht PKCα nd/or PKCε re involved in this regultion. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

21 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Acknowledgements We thnk Miss Din Bkjsov (UAMS) for her help with the mesurements of oxygen consumption nd Dr. Thoms W. Petry (Upjohn Phrmci, Klmzoo, MI) for his generous gift of S(1,2-dichlorovinyl)-L-cysteine. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

22 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. REFERENCES Ae MK, Krth S, Krpov AY, Li J, Lin PT, Kuo WL nd Hershenson MB (1998) Hydrogen peroxide ctivtes extrcellulr signl-regulted kinse vi protein kinse C Rf-1 nd MEK1. Am J Respir Cell Mol Biol 18: Alerti P, Brdell L nd Comolli R (1993) Riosoml protein S6 kinse nd protein kinse C ctivtion y epiderml growth fctor fter temporry renl ischemi. Nephron 64: Anders MW (1995) Mitochondril ioctivtion of cysteine S-conjugte nd 4-thilknotes: Implictions for mitochondril dysfunction nd mitochondril diseses. Biochem Biophys Act 1271:51-57 Blig R, Ued N, Wlker PD nd Shh SV (1997) Oxidnt mechnisms in toxic cute renl filure. Am J Kidney Dis 29: Bertorello A nd Aperi A (1989) N + -K + -ATPse is n effector protein for protein kinse C in renl proximl tuule cells. Am J Physiol 256:F370-F373 Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 Borkn SC nd Schwrtz JH (1989) Role of oxygen free rdicl species in in vitro models of proximl tuule ischemi. Am J Physiol 257:F114-F125 Chen Y, Ci J, Anders MW, Stevens JL nd Jones DP (2001) Role of mitochondril dysfunction in S- (12-dichlorovinyl)-L-cysteine-induced poptosis. Toxicol Appl Phrmcol 170:

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25 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Lsh LH, Hueni SE nd Putt DA (2001) Apoptosis, necrosis nd cell prolifertion induced y S-(1,2- dichlorovinyl)-l-cysteine in primry cultures of humn proximl tuulr cells. Toxicol Appl Phrmcol 177:1-16 Moore RB nd Green T (1988) The synthesis of nephrotoxin conjugtes of glutthione nd cysteine. Toxicol Environ Chem 17: Nony PA, Nowk G nd Schnellmnn RG (2001) Collgen IV promotes repir of renl cell physiologicl functions fter toxicnt injury. Am J Physiol 281:F443-F453 Nony PA nd Schnellmnn RG (2001) Interctions etween collgen IV nd collgen-inding integrins in renl cell repir fter sulethl injury. Mol Phrmcol 60: Nowk G nd Schnellmnn RG (1996) L-scoric cid regultes growth nd metolism of renl cells: improvements in cell culture. Am J Physiol 271:C2072-C2080 Nowk G nd Schnellmnn RG (1997) Renl cell regenertion following oxidnt exposure: inhiition y TGF-β 1 nd stimultion y scoric cid. Toxicol Appl Phrmcol 145: Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 Nowk G, Aleo MD, Morgn JA nd Schnellmnn RG (1998) Recovery of cellulr functions following oxidnt injury. Am J Physiol 274:F509-F515 Nowk G, Kesler KB, McKeller DE nd Schnellmnn RG (1999) Differentil effects of EGF on repir of cellulr functions fter dichlorovinyl-l-cysteine-induced injury. Am J Physiol 276:F228-F236 25

26 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Nowk G, Crter CA nd Schnellmnn RG (2000) Ascoric cid promotes recovery of cellulr functions following toxicnt-induced injury. Toxicol Appl Phrmcol 167:37-45 O Brin CA, Wrd NE, Weinstein IB, Bull AW nd Mrnett LJ (1988) Activtion of rt rin protein kinse C y lipid oxidtion products. Biochem Biophys Res Comm 155: Plumo EJ, Swett JD, Chen S nd Klnn E (1992) Oxidtion-induced persistent ctivtion of protein kinse C in hippocmpl homogentes. Biochem Biophys Res Comm 187: Schnellmnn RG (1988) Mechnisms of t-utyl hydroperoxide-induced toxicity to rit renl proximl tuules. Am J Physiol 255:C28-C33 Sponsel HT, Breckon R nd Anderson RJ (1995) Adenine nucleotide nd protein kinse C regultion of renl tuulr epithelil cell wound heling. Kidney Int 48:85-92 Tessitore L, Perletti GP, Sesc E, Pni P, Piccinini F nd Dinzni MU (1995) Protein kinse C isozymes during liver regenertion Biochem Biophys Res Comm 214: Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 Tock FG (1992) Regenertion fter cute tuulr necrosis. Kidney Int 41: Vmvks S, Bittner D, Deknt W nd Anders MW (1992) Events tht precede nd tht follow S-(1,2- dichlorovinyl)-l-cysteine-induced relese of mitochondril C 2+ nd their ssocition with cytotoxicity to renl cells. Biochem Phrm 44:

27 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. vn de Wter B, Zoeteweij JP, de Bont HJGM, Mulder GJ nd Ngelkerke JF (1994) Role of mitochondril C 2+ in the oxidtive stress-induced dissiption of the mitochondril memrne potentil. J Biol Chem 269: Ysud I, Kishimoto A, Tnk S, Toming M, Skuri A nd Nishizuk Y (1990) A synthetic peptide sustrte for selective ssy of protein kinse C. Biochem Biophys Res Commun 166: Zhng G, Ichimur T, Mier JA, Mcig T nd Stevens JL (1993) A role for firolst growth fctor type-1 in nephrogenic repir. Autocrine expression in rt kidney proximl tuule epithelil cells in vitro nd in the regenerting epithelium following nephrotoxic dmge y S-(1,1,2,2-tetrfluoroethyl)- L-cysteine in vivo. J Biol Chem 268: Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

28 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. Footnotes This work ws supported y Ntionl Institutes of Helth NIDDK Grnt R01 DK59558 nd Pilot Study Grnt from Intrmurl Grnt Progrms t the University of Arknss for Medicl Sciences. Address for reprint requests: Dr. Grzyn Nowk, Deprtment of Phrmceuticl Sciences, University of Arknss for Medicl Sciences, 4301 West Mrkhm St. MS 522-3, Little Rock, AR Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18,

29 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. FIGURE LEGENDS Figure 1. Time-dependent chnges in totl PKC ctivity in cytosolic (A) nd prticulte (B) frctions of RPTC following TBHP-induced injury (0.2 mm; 45 min). Smples of RPTC were collected t different time points fter TBHP exposure nd PKC ctivity mesured in cytosolic nd prticulte frctions s descried in the Methods. Dt re presented s mens + SE, n = 4 seprte RPTC cultures. * P<0.05, significntly different from controls. Figure 2. Time-dependent chnges in totl PKC ctivity in cytosolic (A) nd prticulte (B) frctions of RPTC following DCVC-induced injury (0.2 mm; 90 min). Smples of RPTC were collected t different time points fter DCVC exposure nd PKC ctivity mesured in cytosolic nd prticulte frctions s descried in the Methods. Dt re presented s mens + SE, n = 5 seprte RPTC cultures. * P<0.05, significntly different from controls. Figure 3. The effect of PKC inhiition (Clphostin C, 100 nm) on the recovery of overll mitochondril function in RPTC following TBHP-induced injury (0.2 mm; 45 min). Dt re presented s mens + SE, n = 3 seprte RPTC cultures. Vlues with dissimilr superscripts on given dy re significntly different (P<0.05) from ech other. Figure 4. The effect of PKC inhiition (myristoylted protein kinse C (20-28) peptide derived from pseudosustrte sequence of PKC, 20 µm) on the recovery of overll mitochondril function (A), Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 oxidtive phosphoryltion (A), nd electron trnsport rte (C) in RPTC following TBHP-induced injury (0.2 mm; 45 min). Dt re presented s mens + SE, n = 3 seprte RPTC cultures. Vlues with dissimilr superscripts on given dy re significntly different (P<0.05) from ech other. Figure 5. The effect of PKC ctivtion (PMA, 100 nm) on protein levels of PKCα nd PKCε in cytosolic nd prticulte frctions of RPTC (A) nd phosphorylted nd totl protein levels of PKCα nd PKCε in totl cell homogentes following 5 minute exposure of RPTC to 100 nm PMA (B). C controls, E RPTC treted with diluent (0.1% ethnol), P RPTC treted with 100 nm PMA for 5 29

30 JPET Fst Forwrd. Pulished on Mrch 28, 2003 s DOI: /jpet This rticle hs not een copyedited nd formtted. The finl version my differ from this version. minutes. C. The effect of PKC ctivtion (PMA, 100 nm) on the recovery of overll mitochondril function in RPTC following TBHP-induced injury. D. The effect of PKC ctivtion (PMA, 100 nm) on the recovery of overll mitochondril function in RPTC following DCVC-induced injury. Dt re presented s mens + SE, n = 3 seprte RPTC cultures. Vlues with dissimilr superscripts on given dy re significntly different (P<0.05) from ech other. Figure 6. Decrese in the recovery of ctive N + trnsport y inhiition of PKC in TBHP-injured RPTC. Two dissimilr inhiitors of PKC were used in these studies: A. Myristoylted protein kinse C (20-28) peptide, 20 µm nd B. Clphostin C, 100 nm. Dt re presented s mens + SE, n = 3 seprte RPTC cultures. Vlues with dissimilr superscripts on given dy re significntly different (P<0.05) from ech other. Figure 7. Promotion of the recovery of ctive N + trnsport y PKC ctivtion (PMA, 100 nm) in TBHP-injured (A) nd DCVC-injured (B) RPTC. Dt re presented s mens + SE, n = 3 seprte RPTC cultures. Vlues with dissimilr superscripts on given dy re significntly different (P<0.05) from ech other. Figure 8. Decrese in the recovery of N + -dependent glucose uptke y inhiition of PKC (clphostin c, 100 nm) in TBHP-injured RPTC (A). Lck of n effect of PKC ctivtion (PMA, 100 nm) on the recovery of N + -dependent glucose uptke in TBHP- nd DCVC-injured RPTC (B). Dt re presented s mens + SE, n = 3 seprte RPTC cultures. Vlues with dissimilr superscripts on given dy re Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 18, 2018 significntly different (P<0.05) from ech other. 30