Deletion of Protein Kinase C- Signaling Pathway Induces Glomerulosclerosis and Tubulointerstitial Fibrosis In Vivo
Matthias Meier*,
Jan Menne*,,
Joon-Keun Park*,
Marcel Holtz*,
Faikah Gueler*,
Thorsten Kirsch*,
Mario Schiffer*,
Michael Mengel*,
Carsten Lindschau*,
Michael Leitges* and
Hermann Haller*
* Department of Nephrology, Hannover Medical School, and Phenos GmbH, Hannover, Germany
Address correspondence to: Dr. Matthias Meier, Department of Nephrology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. Phone: +49-0-511-5326319; Fax: +49-0-511-552366; E-mail: meier.matthias{at}mh-hannover.de
Received for publication July 8, 2005.
Accepted for publication February 6, 2007.
Protein kinase C (PKC), a family of 12 distinct serine-threoninekinases, is an important intracellular signaling pathway involvedin various cellular functions, such as proliferation, hypertrophy,apoptosis, and adhesion. PKC-, a novel PKC isoform that is activatedin the diabetic kidney, has been demonstrated to have a centralrole in the underlying signaling infrastructure of myocardialischemia and hypertrophy. The renal phenotype of PKC-–/–mice was studied with regard to renal hypertrophy and fibrosis.PKC-–/– deficient knockout mice were generated andthen killed after 6, 16, and 26 wk of life. Kidney/body weightratio did not show any significant group difference comparedwith appropriate wild-type controls. Urinary albumin/creatinineratio remained normal in wild-type mice, whereas PKC-–/–mice after 6 and 16 wk showed elevated albuminuria. Masson-Goldnerstaining revealed that tubulointerstitial fibrosis and mesangialexpansion were significantly increased in PKC-–/–mice. However, this profibrotic phenotype was not observed inother organs, such as liver and lung. Immunohistochemistry ofthe kidneys from PKC-–/– mice showed increased renalfibronectin and collagen IV expression that was further aggravatedin the streptozotocin-induced diabetic stress model. Furthermore,TGF-1, phospho-Smad2, and phospho-p38 mitogen-activate proteinkinase expression was increased in PKC-–/– mice,suggesting a regulatory role of PKC- in TGF-1 and its signalingpathway in the kidney. These results indicate that deletionof PKC- mediates renal fibrosis and that TGF-1 and its signalingpathway might be involved. Furthermore, these data suggest thatactivation of PKC- in the diabetic state may rather representa protective response to injury than be a mediator of renalinjury.
The family of protein kinase C (PKC) includes at least 12 isoformsthat can be divided in three subgroups, namely classical PKC(PKC-, PKC-1, PKC-2, and PKC-), novel PKC (PKC-, PKC-, PKC-,and PKC-), and atypical PKC (PKC- and PKC-/) (1). Each PKC isoformis a separate gene product, with the exception of PKC-1 and-2, which are alternative spliced variants of the same gene(1). PKC has been identified as the cellular receptor for thelipid second messenger diacylglycerol (DAG) and is thereforea key enzyme in the signaling mechanisms by activation of receptorsthat are coupled to phospholipase C, which leads to a transientelevation in DAG levels (1). Furthermore, PKC is a high-affinityreceptor for phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate(1). PKC isoforms display a distinct cell- and tissue-type expressionpattern or intracellular localization that is considered forthe multiplicity of the PKC isoforms (2,3). In particular, ithas been demonstrated that the activation of PKC increases theproduction of the extracellular matrix (ECM) and various cytokines;enhances contractility, permeability, and vascular cell proliferation;induces the activation of cytosolic phospholipase A2; and inhibitsNa-+K+-ATPase (4,5). All of these events are important featuresof cardiovascular diseases such as diabetes, oxidative stress,and hypertension (4,5). Therefore, PKC is considered to playa pivotal role in intracellular signal modulation and interactionof metabolic and hemodynamic factors that lead to cardiovasculardisease (4,5).
Murine knockout (KO) models of various PKC isoforms now representan exciting tool for studying in vivo effects of such single-genedeletions and how they contribute to the development of cardiovasculardiseases such as diabetes and hypertension (6,7). We recentlyshowed that deletion of PKC-in vivo leads to protection againstthe development of albuminuria in streptozotocin (STZ)-induceddiabetic mice, indicating a specific role of this classicalisoform in the breakdown of the glomerular filtration barrierin the diabetic state (8). It is interesting that we failedto demonstrate a role for this PKC isoform in the developmentof TGF-1–mediated renal hypertrophy in this KO mouse model(8). However, Klein et al. (9) recently demonstrated that deletionof PKC-, a novel PKC isoform that is postulated to be involvedin the regulation of myocardial ischemia-reperfusion, leadsto increased murine myocardial fibrosis in a chronic pressureoverload model that is associated with diastolic dysfunction.Furthermore, a PKC-–dependent pathway was recently proposedin the development of interstitial lung fibrosis in patientswith systemic sclerosis (10). These results implicate a rolefor PKC- in the development of other fibrotic diseases suchas chronic renal disease (e.g., diabetic nephropathy). In thisstudy, we therefore investigated the functional role of PKC-in renal physiology by means of PKC-–/– KO miceand tested the hypothesis that PKC- is involved in chronic renalfibrosis in the STZ-induced diabetic mouse model.
Animal Studies
Experiments were performed with male hybrid C57BL/6 x 129/Olawild-type (WT) littermates and PKC-–/– KO mice (9,11).PKC-–/– mice were developed as described elsewhereand published previously (9,11). In brief, embryonic day 14stem (ES) cells (129/Ola) were used for the targeting experiment.Homologue-recombined ES cell clones were then introduced byinjection into C57BL/6 blastocytes. The possible germline transmissionof the injected ES cells was identified by crossing the observedchimeric males to C57BL/6 females and subsequently the presenceof agouti coat color in the F1 progeny. F1 heterozygote breedinggave rise to homozygote mice, which were finally used in thisstudy and correspond to the hybrid (C57BL/6 x 129/Ola) background.KO of the PKC- gene in the individual KO mice was confirmedusing real-time PCR. The mice received a standard diet withfree access to tap water. All procedures were carried out accordingto guidelines from the American Physiologic Society and wereapproved by local authorities.
To induce a diabetic state in the rodents, 7-wk-old weight-matchedmice received either 50 mg/kg body wt STZ (Sigma-Aldrich, Munich,Germany) in 50 mM sodium citrate (pH 4.5) or sodium citratebuffer intraperitoneally on five consecutive days. Blood glucosemeasurement was performed from tail blood using the glucometerElite (Bayer, Leverkusen, Germany) every other day. Mice withglucose levels >15 mmol/L on two consecutive measurementswere regarded as hyperglycemic, and glucose measurements wereextended to once weekly. Mice that had no hyperglycemia 20 dafter the last STZ injection were not used for our experiments.The mice received no insulin within the complete study period.Ketonuria did not occur (data not shown). After 8 wk of hyperglycemia,the mice were killed according to a standardized protocol aspreviously published (8). Briefly, after anesthesia with Avertin(2.5%), a laparotomy was performed and urine was collected bypuncturing the bladder with a 23-G needle. Then, the abdominalaorta was cannulated again with a 23-G needle and the organswere perfused with lactated Ringer solution. After ligationof the left renal artery, the left kidney was removed, weighed,and snap-frozen in isopentane (–40°C). Afterward,the right kidney was perfused with 3% paraformaldehyde in 0.1M Sörensen's phosphate buffer. The right kidney was fixedfor an additional 20 h in 3% paraformaldehyde in Sörensen'sphosphate buffer and then paraffin embedded.
Cell Culture of Isolated Vascular Smooth Muscle Cells
Aortic vascular smooth muscle cells (VSMC) were prepared fromWT and PKC-–/– mice and cultured as previously published(12). The differentiated phenotype of the cultured VSMC wasdetermined by staining cells for -actin and desmin. Cells werewashed with ice-cold PBS and scraped off on ice. Cell lysiswas done by boiling the cell pellet in lysis buffer (50 mM TrisHCl [pH 7.4], 2% SDS, 1 mM EDTA, and 1 mM EGTA). After shorthigh-speed centrifugation (5 min, 15,000 x g), protein concentrationof the supernatant was estimated by the Bradford method and30 µg of total protein was loaded in each lane. Nonreducingconditions resulted in dimer formation of TGF-1 as proved incontrol experiments. Quantification was done with Scion Image(Frederick, MD) by correction for loading differences usinga housekeeper protein (-actin).
Urine Analysis
Albumin concentration in spot urine samples was measured witha commercially available competitive ELISA following the instructionsof the manufacturer (Exocell, Philadelphia, PA) and was normalizedto urine creatinine defined as albumin/creatinine ratio. Urinecreatinine was measured with the Beckman Creatinine Analyzer(Beckman Instruments, Fullerton, CA) using a modified Jafféreagent. The rate of increase in absorbance as a result of theformation of alkaline creatinine picrate complex was measured.The rate of complex color formation was directly proportionalto the concentration of the creatinine in the sample.
Morphometric Studies
For morphologic evaluation, 3-µm paraffin sections werestained with trichrome Masson-Goldner standard procedure (8).Immunohistochemistry was performed using the following primaryantibodies: TGF-1 (sc-146; Santa Cruz Biotechnology, Santa Cruz,CA), fibronectin (14-109-0568; Paesel+Lorei, Frankfurt, Germany),type IV collagen (1340-01; Southern Biotechnology, Birmingham,AL), phospho-p38 mitogen-activated protein kinase (phospho-p38MAPK; 9211; Cell Signaling, Danvers, MA), and phospho-Smad2(3101; Cell Signaling). For indirect immunofluorescence, nonspecificbinding sites were blocked with 10% normal donkey serum (JacksonImmunoResearch, West Grove, PA) for 30 min. Then cryosectionswere incubated with the primary antibody for 1 h. All incubationswere performed in a humid chamber at room temperature. For fluorescencevisualization of bound primary antibodies, sections were furtherincubated with Cy3-conjugated secondary antibodies (JacksonImmunoResearch) for 1 h. Specimens were analyzed using a ZeissAxioplan-2 imaging microscope with the computer program AxioVision3.0 (Zeiss, Jena, Germany).
Western Blotting
Frozen kidneys were pulverized in liquid nitrogen and resuspendedin 2 ml of lysis buffer (20 mM Tris buffer [pH 7.5] that contained10 mM glycerol-phosphate, 2 mM pyrophosphate, 1 mM sodium fluoride,1 mM PMSF, 1 g/ml leupeptin, 1 mM dithiothreitol, and 1 mM EDTA)as described previously (8). Homogenates were sonicated forthree 20-s bursts on ice and centrifuged at 500 x g for 1 minto remove cell debris. Aliquots of the supernatants were storedat –80°C. The protein amount was measured using theLowry assay. A total of 70 µg of protein of each samplewas suspended in loading buffer and run on a 10% polyacrylamidegel and electrophoretically transferred to nitrocellulose membrane.Membranes were blocked in 5% skim milk and 1% BSA for 1 h atroom temperature. Primary antibody against factor TGF-1 (sc-146;Santa Cruz Biotechnology) was applied with gentle rocking overnightat 4°C. After three, 10-min washing steps with TBST buffer(50 mmol/L Tris HCl [pH 7.5], 150 mmol/L NaCl, and 0.01% Tween20), incubation with horseradish peroxidase–conjugatedgoat anti-rabbit secondary antibody (Dianova, Hamburg, Germany)was performed for 1 h at room temperature. After three additionalTBST washes, the membrane was incubated with Renaissance reagent(NEN Life Science, Zaventem, Belgium) according to the manufacturer'sinstructions and exposed to x-ray film (Kodak, Stuttgart, Germany).Quantitative analysis was done by measuring relative density(Scion Image).
Confocal Microscopy
Confocal microscopy was performed as described previously (9).The preparation was mounted with medium (Aqua Polymount; Polyscience,Warrington, PA) under a glass coverslip and analyzed with aNikon-Diaphot microscope. A confocal imaging system (Bio-RadMRC 1024, Bio-Rad Laboratories, Munich, Germany) with a ultravioletsource and an argon/krypton laser was used. At least 30 cellsfrom each of at least three independent experiments were examinedunder each experimental condition. For each set of experiments,identical settings for power of the light source, confocal aperture,gain, and black level were used. Quantification of the signalintensity of single cells was done with histogram/area functionsin the available software (Lasersharp or NIH Image). The cellswere outlined manually, and the mean fluorescence intensitywas obtained for the delineated regions. Data are presentedas relative fluorescence intensity.
Statistical Analyses
The data were compared by ANOVA and the unpaired t test as appropriate,and values are displayed as mean ± SEM. Significant differenceswere accepted at P < 0.05. Data analysis was performed usingSPSS 11.0 software (SPSS, Chicago, IL).
We started our investigation of PKC-–/– (KO) miceat the ages of 6, 16, and 26 wk in comparison with appropriateWT littermates to analyze changes of the renal phenotype overtime. All mice were screened for clinical parameters such asbody and kidney weight with subsequent calculation of the kidney/bodyweight ratio. Furthermore, the albumin/creatinine ratio wasmeasured in spot urine that was sampled before the mice werekilled and kidneys were removed. Subsequent morphometric studiesof the kidney tissue were performed using Masson-Goldner stainingto evaluate structural renal changes in both WT and PKC-–/–mice.
Clinical data of baseline (week 6, day 42), intermediate (week16, day 112), and final (week 26, day 168) time points are displayedin Tables 1 and 2. The total body weight was comparable in WTand KO mice but increased significantly over time in both groupsto a similar extent because of general growth (Table 1). Furthermore,kidney weight also increased with age (Table 2), so the calculatedkidney/body weight ratio remained stable over time. Notably,no significant group differences between WT and KO groups wereobserved during the entire study period. Importantly, the kidney/bodyweight ratio in the PKC-–/– mice was comparableto that of the WT controls at all three time points (Table 2).These results demonstrate that PKC-–/– mice didnot show renal hypertrophy and that PKC- signaling does notinterfere with renal growth.
Table 1. Body weight and albumin/creatinine ratio of baseline (day 42, week 6; n = 5), intermediate (day 112, week 16; n = 13), and final (day 168, week 26; n = 10) groupsa
Table 2. Kidney weight and kidney/body weight ratio of baseline (day 42, week 6; n = 5), intermediate (day 112, week 16; n = 13), and final (day 168, week 26; n = 10) groupsa
It is interesting that urinary albumin/creatinine ratio wassignificantly increased in PKC-–/– KO mice after16 wk (30.0 ± 10.2 versus 3.8 ± 1.3 g/mol; P <0.01). The baseline group after 6 wk already showed a tendencytoward elevated albuminuria compared with appropriate WT controls(25.9 ± 21.9 versus 3.6 ± 2.4 g/mol; P > 0.05),although data variability between the individual mice was veryhigh (Table 1). However, after 26 wk, we did not see any differencewith regard to the urinary albumin/creatinine ratio betweenKO and WT mice. These results suggest that deletion of PKC-may exert functional or structural changes in the kidney thatmay not lead to consistent albuminuria in this KO model. Therefore,we subsequently performed tissue analysis of mouse kidneys fromWT and KO mice by means of light microscopy to evaluate possiblestructural changes in PKC-–/– mice. Notably, weobserved progressively increased renal fibrosis in all PKC-–/–groups over time as shown in Figure 1. Masson-Goldner stainingrevealed increased tubulointerstitial fibrosis and glomerulosclerosisin the KO mice (Figure 1, top) compared with the appropriateWT control mice (Figure 1, bottom) at the ages of 6, 16, and26 wk. Figure 1, top, displays already increased tubulointerstitialfibrosis as displayed as more pronounced blue staining in 6-wk-oldKO mice (Figure 1A) which is further aggravated in the KO groupsat the age of 16 (Figure 1B) and 26 wk (Figure 1C) comparedwith appropriate WT controls (Figure 1, D through F). In conclusion,the observed profibrotic renal phenotype of PKC-–/–explains the increased albuminuria that was observed in theclinical examination reported previously. However, renal fibrosisis more pronounced than albuminuria in PKC-–/– mice.
Figure 1. Light microscopy of the kidney after trichrome (Masson-Goldner) staining reveals increased tubulointerstitial fibrosis and glomerulosclerosis in protein kinase C-–deficient (PKC-–/–) mice with age. (Top) Morphologic changes of PKC-–/– mice with increased blue staining of the profibrotic changes at the ages of 6 (A), 16 (B), and 26 wk (C) compared with appropriate wild-type (WT) mice (D through F, respectively).
We then extended our morphometric studies to other organs thatare susceptible to fibrosis in humans, mainly liver and lung.In contrast to the observed changes in the kidneys from PKC-–/–,light microscopy of liver tissue did not show any significantfibrosis in KO mice compared with WT controls, whereas lungtissue exhibits slightly increased pulmonary fibrosis in theKO mice. Figure 2 displays representative Masson-Goldner stainsof liver and elastic van Gieson stains of lung tissue from 26-wk-oldPKC-–/– mice. The latter demonstrates only a minorincrease of pulmonary fibrosis in the PKC-–/– mice.The predominant profibrotic phenotype that was observed in thekidneys from PKC-–/– mice therefore represents organspecificity to the kidney rather than a systemic effect. Thisconclusion goes ahead with the observation that the generallife span of this KO breed is not abbreviated. Furthermore,these data suggest that stimulated PKC- signaling might be ableto reduce or prevent chronic (murine) kidney disease but doesnot seem to be critical in other organ systems that are proneto fibrosis.
Figure 2. In contrast to the observed changes in kidneys of PKC-–/– mice, light microscopy of liver and lung tissue did not show any fibrosis in the knockout (KO) mice. Representative pictures from liver (A) and lung (B) of 26-wk-old PKC-–/– mice are displayed.
Because PKC isoform specificity is of considerable importancein vascular complications in diabetes mellitus (4) and becausewe and others previously showed that the expression and activationin PKC- and - are increased in the diabetic rat kidney (13,14),we next studied the role of PKC- signaling in chronic renaldisease such as diabetic nephropathy. We tested the hypothesisthat deletion of PKC- exacerbates experimental diabetic nephropathy,mainly renal fibrosis, in the STZ-induced diabetic mouse model.Hyperglycemia was induced in 7-wk-old mice by intraperitonealinjection of STZ (50 mg/kg body wt) on days 1 through 5. Bloodglucose levels >15 mmol/L were achieved within 20 d afterthe last STZ injection and persisted in both WT littermatesand PKC-–/– (KO) mice, diabetic groups as comparedwith the appropriate, sham-injected control mice. After 8 wkof diabetes, urinary albumin/creatinine ratio was significantlyincreased in diabetic KO mice compared with healthy and diabeticWT mice (44.9 ± 11.4 versus 3.8 ± 1.3 [P <0.01], respectively; 12.0 ± 1.6 mmol/L [P < 0.05])and further increased compared with nondiabetic PKC-–/–mice (30.0 ± 10.2 mmol/L). These data indicate that thischronic diabetic stress model is able to exacerbate and outlinefurther the previously demonstrated renal changes in the PKC-–/–mice. Second, no group difference regarding kidney/body weightratio was observed between the nondiabetic and diabetic KO mice(8.94 ± 0.7 versus 8.60 ± 1.1; P > 0.05), indicatingalso no increased renal hypertrophy in the diabetic state inPKC-–/– mice. Masson-Goldner staining revealed amore pronounced tubulointerstitial fibrosis and glomerulosclerosisin diabetic KO mice compared with appropriate WT control mice(Figure 3). These results suggest that the increased expressionand activation of the PKC- isoform that previously was shownin experimental diabetic nephropathy (13,14) does not induceprofibrotic changes in the (diabetic) kidney but rather maintainsantifibrotic functions even in the nondiabetic kidney.
Figure 3. Streptozotocin (STZ)-induced diabetes (8 wk diabetes duration) leads to further exacerbated tubulointerstitial fibrosis (top) and glomerulosclerosis (bottom) in PKC-–/– mice in light microscopy after Masson-Goldner staining. KO mice (B) displayed increased tubulointerstitial fibrosis compared with WT controls under diabetic condition (A). Comparable results are also observed on the glomerular level in the diabetic state (WT control [C] and KO mice [D]).
We then investigated matrix expansion by means of immunohistochemistryof ECM proteins to evaluate further the molecular mechanismsthat lead to the profibrotic phenotype of PKC-–/–compared with WT control mice at the age of 16 wk. Figure 4,top, displays increased tubulointerstitial expression of fibronectinin KO mice compared with WT mice. Similar expression patternsare also shown in Figure 4, bottom, where mesangial expressionof collagen IV is shown. These results clearly indicate thatPKC- signaling is directly involved in profibrotic mechanisms,such as ECM expansion, that are observed in chronic renal diseases.
Figure 4. Immunohistochemistry of renal tissue from PKC-–/– KO mice shows significantly increased tubulointerstitial and mesangial matrix expression. Fibronectin (top) staining displayed increased tubulointerstitial expression in KO (B) compared with WT (A) control mice. Similar expression patterns are also shown on the mesangial level (bottom) using collagen IV staining in the appropriate study groups (WT control [C] and KO mice [D]).
TGF-1 represents a key cytokine that mediates renal hypertrophyand accumulation of ECM in chronic renal disease such as diabeticnephropathy (4,12). For further extrapolation of how deletionof PKC- leads to increased glomerulosclerosis and tubulointerstitialfibrosis, immunohistochemistry of TGF-1 was subsequently performed.We revealed that TGF-1 expression is increased in PKC-–/–mice compared with WT controls (Figure 5). These changes mainlyoccur on the glomerular level (Figure 5, bottom) where, again,the KO mice displayed a significantly higher expression of TGF-1than in controls. Figure 5, top, displays increased tubulointerstitialTGF-1 staining of WT and PKC-–/– mice at the ageof 16 wk as well.
Figure 5. Immunohistochemistry of expression. KO mice (B and D) displayed a significantly increased TGF-1 expression, mainly on the glomerular level, compared with WT controls (A and C).
To study quantitatively the increase of TGF-1 protein expression,we next performed Western blot analysis (Figure 6A) and confocalmicroscopy (Figure 6B) in VSMC that were isolated from WT andPKC-–/– mice. Furthermore, cells were starved undernormal as well as high-glucose conditions for 16 h to outlinethe phenotype change. Again, quantitative analysis revealedthat VSMC from KO mice showed significantly increased TGF-1expression compared with VSMC from WT mice, which is furtherstimulated by the high-glucose condition. These results indicatethat the TGF-1 signaling pathway is modified by the deletionof PKC-.
Figure 6. Quantitative protein analysis of TGF-1 confirmed increased expression levels in PKC-–/– mice using Western blot (A) and confocal microscopy (B). (A) Increased protein expression of TGF-1 under high-glucose condition in cultured vascular smooth muscle cells (VSMC) from KO mice compared with VSMC from WT controls. (B) Confocal microscopy of TGF-1 expression in cultured VSMC from WT (top) and KO mice (bottom). Comparable to the observed changes in vivo, the most abundant expression level of TGF-1 was found in the VSMC from PKC–/– KO mice that were starved under high-glucose condition.
We also studied whether deletion of PKC- signaling not onlyleads to increased expression of TGF-1 but also induces activationof the TGF-1 signaling pathway (Figure 7). We therefore performedimmunohistochemistry of kidneys from 16-wk-old WT and KO miceto study phospho-Smad-2 expression. The results clearly demonstratedincreased expression of phospho-Smad2 in PKC-–/–mice, proving activation of the TGF-1 receptor signaling pathwaywhen PKC- is deleted (Figure 7, top). Furthermore, immunohistochemistryusing a specific phospho-antibody against p38 MAPK (Figure 7,bottom), another signaling cascade downstream of the TGF-1 receptorthat is postulated to be involved in cellular processes suchas growth and differentiation (15), was subsequently performed.Again, increased phospho-Smad-2 and phospho-p38 MAPK expressionis further increased when diabetes mellitus is induced in thePKC-–/– mice (data not shown). Deletion of PKC-was shown to activate the p38 MAPK signaling pathway mainlyin the glomeruli. These results indicate that the enhanced signalingof this inflammatory pathway might be an important mechanisminvolved in the increased glomerulosclerosis and tubulointerstitialfibrosis in the PKC- KO mice.
Figure 7. TGF-1 signaling pathway is activated in PKC-–/– mice. Immunohistochemistry of phospho-Smad2 (top) and phospho-p38 mitogen-activated protein kinase (phospho-p38 MAPK; bottom) revealed increased activation of the TGF-1 signaling pathway compared with nondiabetic (A and E) and diabetic (C and G) controls when PKC- is deleted (B and F). Again, in the diabetic state, the activation level is further increased (D and H).
In this study, we investigated the functional role of PKC- inrenal physiology by means of PKC-–/– KO mice andtested the hypothesis that PKC- is involved in chronic renalfibrosis in the STZ-induced diabetic mouse model. It is interestingthat the results demonstrate an organ-specific renal phenotypein PKC-–/– mice with increased tubulointerstitialfibrosis and glomerulosclerosis. Deletion of PKC- signalingthereby might interfere with activation of the TGF-1 pathway.The diabetic stress model was able further to enhance renalfibrosis and outline albuminuria in the PKC-–/–mice. These data suggest that the previously reported increasedexpression and activation of PKC- represents a protective responseto injury rather than a mediator of renal injury in diabetes,as has been demonstrated for the classical PKC isoforms and (4,5,8,12–14,16,17).
PKC-, a novel PKC isoform that is characterized as calcium-independentand phorbol ester/DAG-sensitive serine/threonine kinase, isexpressed in many tissues in cell types (18). To date, the essentialroles of PKC- have been established in many signaling systems,including proliferation, differentiation, gene expression, musclecontraction, mechanical force adaptation, metabolism, transport,exocytosis, and endocytosis, and also have nervous, inflammatory,immune, and circular functions (18). Recent work from Redlinget al. (7) revealed that the PKC- isoform is mainly expressedin glomeruli, proximal convoluted tubules, and medullary thickascending limbs in the mouse kidney, whereas its expressionprofile in the rat kidney is less consistent. We previouslyshowed that PKC- expression was mainly increased in renal tubulesin diabetic rats but only weakly expressed in the glomeruliof STZ-induced diabetic rat kidneys (13). This result was confirmedby Ha et al. (19), who also demonstrated enhanced translocationof PKC- and - mainly in diabetic tubules from rat kidney. Incontrast, Whiteside et al. (14) suggested that PKC- activationalso contributes to glomerular changes in early diabetic ratnephropathy.
In this study, we observed increased tubulointerstitial fibrosisand glomerulosclerosis in PKC-–/– mice, whereasa systemic profibrotic phenotype was not observed. We did notsee increased liver fibrosis and observed only slightly increasedlung fibrosis in PKC-–/– mice. However, a PKC-–dependentpathway was recently proposed in the development of interstitiallung fibrosis in patients with systemic sclerosis (10,20,21).It has been demonstrated that the regulation of an ECM molecule(tenascin-C) is controlled by the PKC- isoform in lung fibroblastsfrom patients with systemic sclerosis (21). In addition, thesignaling pathway involving PKC- and phase 2 detoxificationenzymes heme oxygenase 1 and glutathione S-transferase P1, isdefective, leading to decreased PKC- levels in scleroderma lungfibroblasts in vitro and in fibrotic lung tissue in vivo (10).Furthermore, PKC- plays critical roles in protecting againstischemic damage, whereas overexpression and activation of PKC-results in myocardial hypertrophy (22,23). In conclusion, ourin vivo data add to the somewhat complex situation that deletionof PKC- leads to organ-specific renal fibrosis.
So far, it is largely accepted that high-glucose–inducedexpression and translocation to the membranous compartment (activation)of PKC isoforms is a key mediator of microvascular complicationsin the diabetic state, leading to subsequent induced expressionand stimulated action of various cytokines such as TGF-1 (4,5,14).Several studies previously showed increased renal expressionand activation of PKC- in STZ-induced experimental diabeticnephropathy in various rodent models, indicating an importantfunctional role of this novel PKC isoform in the developmentof chronic kidney disease (13,18,24,25). We therefore induceddiabetes with STZ in our mouse model, which further enhancedthe profibrotic phenotype in the PKC-–/– mice. Thediabetic state was able to pronounce further tubulointerstitialfibrosis and glomerulosclerosis when PKC- was deleted. Thisresult suggests that increased expression and activation ofPKC-, which was previously demonstrated in experimental STZ-induceddiabetes, represents a protective response to injury ratherthan a mediator of diabetic nephropathy (4,5,8,12–14,16,17).
TGF-1 causes the transformation of interstitial fibroblastsinto myofibroblasts and leads to the overproduction of ECM followedby tubulointerstitial fibrosis (15). Binding of TGF- to itsTGF--RII and subsequent recruitment of the TGF--RI results inphosphorylation and activation of receptor-regulated Smads,a series of transducer proteins that associate to heteromultimerswhen activated (26). The Smad complex then translocates to thenucleus, where it regulates the expression of TGF- target genesthrough direct binding to DNA via binding to Smad-binding elementand/or interaction with other transcription factors (26). Yakymovychet al. (27) demonstrated that Smad2 and Smad3, the targets ofTGF- receptors, can be phosphorylated in their MH1 domain byPKC, indicating potential cross-talk between classical TGF-and PKC signaling cascades. Furthermore, it has been shown thatTGF-1 activates PKC- in human mesangial cells and that thisactivation plays a role in TGF-1–stimulated transcriptionalactivity and collagen I gene transcription (28). PKC, however,is also known to be involved in hyperglycemia-stimulated TGF-1promoter activity in mesangial cells (29). This could be themechanism that leads to increased TGF-1 mRNA expression andprotein synthesis that have been observed in murine mesangialcells that were cultured in high glucose (30).
PKC-–/– mice showed only inconsistent albuminuriacompared with WT controls. However, induction of STZ diabetesmellitus as a chronic stress model led to a significantly increasedurinary albumin/creatinine ratio in PKC-–/– mice.We previously showed that deletion of PKC-in vivo leads toprotection against the development of microalbuminuria in STZ-induceddiabetic mice, indicating a specific role of this classicalisoform in the breakdown of the glomerular filtration barrierin the diabetic state (8). Furthermore, we have shown that highglucose leads to a PKC-–dependent expression of TGF-1in VSMC under in vitro conditions (12) but failed to demonstratea role for PKC- in the regulation of TGF-1 expression in vivoin murine diabetic nephropathy (8). Such controversy resultsmight be explained by the fact that glucose stimulates differentPKC isoforms in various cell types or that short-term activationof PKC isoform by high glucose concentrations in vitro has adifferent cellular effect than the long-term effects of hyperglycemiain vivo. Complex diversity in tissue expression and cellularcompartmentalization that is critical for the activation ofspecific PKC isoforms therefore results in controversial invitro studies (4,5). Thus, murine KO models of various PKC isoformsnow represent an exciting option for studying in vivo effectsof such single-gene deletions and how they contribute to thehigh-glucose–mediated transformation of the kidney intoa sclerotic phenotype (6,8).
Our results suggest a regulatory role of the PKC- isoform inthe development of renal fibrosis and that TGF-1 and its signalingpathway might be involved. Therefore, PKC- activation may alsohave a potential key regulatory function in chronic renal diseasesuch as diabetic nephropathy. However, the detailed molecularmechanism for how deletion of PKC- results in this valuableprofibrotic KO model awaits further studies.
This article was supported by grants from SERVIER-EFSD (EuropeanFoundation for the Study of Diabetes) to M. Meier and the GermanResearch Council (Deutsche Forschungsgemeinschaft/DFG) to H.Haller and J. Menne.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Signaling Pathway Induces Glomerulosclerosis and Tubulointerstitial Fibrosis In Vivo
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Abstract
Introduction
, a novel PKC isoform that is activated in the diabetic kidney, has been demonstrated to have a central role in the underlying signaling infrastructure of myocardial ischemia and hypertrophy. The renal phenotype of PKC-
1, phospho-Smad2, and phospho-p38 mitogen-activate protein kinase expression was increased in PKC-
, PKC-
), novel PKC (PKC-
, PKC-
, and PKC-
), and atypical PKC (PKC-
and PKC-
/
) (1). Each PKC isoform is a separate gene product, with the exception of PKC-
