Calpain Activation and Secretion Promote Glomerular Injury in Experimental Glomerulonephritis: Evidence from Calpastatin-Transgenic Mice
Julie Peltier*,,
Agnès Bellocq*,,,
Joëlle Perez*,
Sophie Doublier,
Yi-Chun Xu Dubois*,,,
Jean-Philippe Haymann*,,,
Giovanni Camussi and
Laurent Baud*,,
* INSERM, U702, Université Pierre et Marie Curie-Paris6, UMRS702, AP-HP, Tenon Hospital, Department of Physiology, Paris, France; and Center of Experimental Research and Medical Sciences, Department of Internal Medicine, University of Turin, Turin, Italy
Address correspondence to: Dr. Laurent Baud, INSERM U702, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France. Phone: +33-1-5601-7951; Fax: +33-1-5601-7003; E-mail: laurent.baud{at}tnn.ap-hop-paris.fr
Received for publication May 31, 2006.
Accepted for publication September 26, 2006.
Glomerular injury and albuminuria in acute glomerulonephritisare related to the severity of inflammatory process. Calpain,a calcium-activated cysteine protease, has been shown to participatein the development of the inflammatory process. Therefore, fordetermination of the role of calpain in the pathophysiologyof acute glomerulonephritis, transgenic mice that constitutivelyexpress high levels of calpastatin, a calpain-specific inhibitorprotein, were generated. Wild-type mice that were subjectedto anti–glomerular basement membrane nephritis exhibitedelevated levels of calpain activity in kidney cortex at theheterologous phase of the disease. This was associated withthe appearance in urine of calpain activity, which originatedpotentially from inflammatory cells, abnormal transglomerularpassage of plasma proteins, and tubular secretion. In comparisonwith nephritic wild-type mice, nephritic calpastatin-transgenicmice exhibited limited activation of calpain in kidney cortexand limited secretion of calpain activity in urine. This wasassociated with less severe glomerular injury (including capillarythrombi and neutrophil activity) and proteinuria. There wasa reduction in NF-B activation, suggesting that calpain mayparticipate in inflammatory lesions through NF-B activation.There also was a reduction in nephrin disappearance from thesurface of podocytes, indicating that calpain activity wouldenhance proteinuria by affecting nephrin expression. Exposureof cultured podocytes to calpain decreased nephrin expression,and, conversely, exposure of these cells to calpastatin preventedTNF- from decreasing nephrin expression, demonstrating a rolefor the secreted form of calpain. Thus, both activation andsecretion of calpains participate in the development of immuneglomerular injury.
Inflammatory damages to the glomerulus characterize acute glomerulonephritis,an immune-mediated disorder that is a major cause of renal failure.The experimental model of anti–glomerular basement membrane(anti-GBM) nephritis has been used widely to analyze the underlyingmechanisms of this inflammatory process (1). Injection of heterologousanti-GBM antiserum to rabbit, rat, or mouse causes immediateand transient, complement-dependent influx of polymorphonuclearneutrophils into glomerular capillaries that is responsibledirectly or indirectly for both proteinuria and glomerular fibrindeposition (heterologous phase) (2). Immune response againstthe heterologous Ig leads later to mononuclear cell recruitmentand subsequent proteinuria (autologous phase).
Calpains are calcium-activated neutral cysteine proteases (3,4).Two major isoforms, calpain µ, or I, which requires micromolarCa2+ concentrations for activity and calpain m, or II, whichrequires millimolar Ca2+ concentrations, are ubiquitously expressed,whereas the other isoforms are tissue-specific forms. Everycalpain isoform is present in the cytosol as an inactive proenzyme.Binding of Ca2+ to µ- or m-calpain induces the releaseof constraints that are imposed by domain interactions and resultsin a two-stage activation process, with first the release ofan approximately 30-kD regulatory subunit and second the rearrangementof the active site cleft in an approximately 80-kD catalyticsubunit (5). Calpain activity is tightly controlled by calpastatin,a specific endogenous inhibitor that contains four equivalentinhibitory domains (3).
Calpains play an important role in the inflammatory process.First, they are involved in the activation of NF-B and, thereby,in the NF-B–dependent expression of proinflammatory cytokinesand adhesion molecules. Underlying mechanisms include degradationof the PEST (proline, glutamate, serine, threonine) sequencein the inhibitor IB, a key step in nuclear translocation ofNF-B (6). Second, calpains are critical for inflammatory celladhesion and chemotaxis and inflammatory mediator processing(7,8). Third, calpains are implicated in the cleavage of theheat-shock protein 90, which is required to maintain glucocorticoidreceptor in a ligand-binding conformation (9). As a consequence,binding and anti-inflammatory efficacy of glucocorticoid arelimited in inflammatory cells. Finally, calpains are externalizedduring the inflammatory process and play a role in the microenvironmentof inflammatory cells (10), for instance by facilitating theirrecruitment (11). Thus, both intracellular and externalizedcalpains would strengthen inflammatory process. The observationthat calpain inhibitor administration decreases inflammatorylesions in lung, joint, and kidney supports this hypothesis(12,13).
Whether calpains are involved in the course of acute glomerulonephritisstill is unknown. Therefore, the aim of this study was to measurethe activity of calpains in kidney tissue and urine of C57BL/6mice with anti-GBM nephritis and to characterize the pathogenicrole of these enzymes. Different approaches could be consideredto hamper calpain activation: First a pharmacologic approach.Second, a disruption of calpain gene; but it is a complex approachbecause different forms of calpains have been identified. Inaddition, disruption of calpain 4 gene, a common regulatorysubunit that is shared by different forms of calpains, completelyblunts calpain activity and results in a lethal phenotype (14).Therefore, we opted for a third approach: To create mice thatare transgenic for calpastatin. Its overexpression affects potentiallythe different forms of calpains, the activity of which is bluntedrather than suppressed.
Cells
Human proximal tubular epithelial cells (HK-2 cell line; AmericanType Culture Collection, Manassas, VA) were cultured at 37°Cin a serum-free keratinocyte medium supplemented with humanrecombinant EGF and pituitary bovine extract (Life Technologies,Cergy Pontoise, France) under a 5% CO2 and 95% air atmosphere.Primary cultures of human podocytes were established and characterizedas described previously (15). Established lines of differentiatedpodocytes were obtained by infection of pure primary cultureswith a hybrid Adeno5/SV40 virus and characterized as describedpreviously (15). They were cultured in DMEM that contained 10%FCS (Euroclone, Wetherby West Yorkshire, UK). Polymorphonuclearneutrophils were isolated from blood of C57BL/6 mice by sedimentation,using Lymphoprep (Axis-Shield PoC AS, Oslo, Norway).
Mice
Studies were conducted in female C57BL/6 mice that weighed 15to 20 g. They were housed in a room with constant temperatureand a 12-h dark/light cycle and fed ad libitum on standard mousefood. All procedures involving these animals were conductedin accordance with national guidelines and institutional policies.Podocin-deficient (Nphs2–/–) mice were providedby C. Antignac (INSERM U574, Hôpital Necker-Enfants Malades,Paris, France).
Calpastatin-transgenic mice were created in the laboratory.To this aim, we obtained the cDNA clone of rabbit calpastatin(PM 194) from Marc Piechaczyk (CNRS UMR 9942). After sequencing,Xho1-Xba1 fragment of this cDNA was inserted on the PCI expressionvector, which includes a viral promoter (cytomegalovirus immediate-earlyenhancer/promoter region). Microinjections were carried outinto fertilized ovocytes of C57BL/6 x DBA2 mice. The presenceand the expression of the transgene were identified in foundertransgenic mice by PCR and reverse transcriptase–PCR (RT-PCR)analysis, respectively. All mice that were used in these studieswere homozygous for the transgene and backcrossed into the C57BL/6background nine generations.
Tail biopsies from F1 mice were genotyped by PCR. Total DNAwas quantified spectrophotometrically at 260 nm and stored at–20°C. Each subsequent PCR (25 µl) contained1 µl of template cDNA, 0.5 µl of dNTP 10 mM, 0.5µl of each primer, 0.7 µl of MgCl2 50 mM, 2.5 µlof 10x PCR buffer, and 0.125 µl of Taqpolymerase (LifeTechnologies). Primers were as follows (5'>3'): 3' GTTGGCTTAGGCTGCTTTTCGTand 5' CCAGACTCCGTGACACCCCTT. PCR cycling was as follows: step1, 4 min at 94°C; step 2, 40 cycles of 45 s at 94°C,1 min at 60°C, and 1 min at 72°C; and step 3, 10 minat 72°C. The PCR products were separated on 1.5% agarosegels, stained with ethidium bromide, and photographed underultraviolet lights.
Expression of the transgene was evaluated by RT-PCR. Total RNAwere extracted from tail fragments, quantified spectrophotometricallyat 260 nm, and stored at –20°C. First-strand cDNAsynthesis was performed with the superscript RNase H- reversetranscriptase kit (Life Technologies) and random hexamer primers.Total RNA (2 µg) was diluted in RNase-free water to 10µl. First-strand buffer (5x; 4 µl), dithiothreitol(0.1 M, 2 µl), dNTP (10 mM, 1 µl), pdN6 (1000 µg/ml,0.5 µl; Amersham, Orsay, France), oligo(dT) 12 to 18 (0.5µg/µl, 0.5 µl; Invitrogen, Cergy Pontoise,France), Rnasin (0.2 µl; Promega, Madison, WI), and RNAse-freewater (0.8 µl) were added to each sample of diluted RNA.One microliter of RT enzyme or RNase-free water were added tothe RT+ and control samples, respectively. The reaction wasallowed to proceed for 10 min at 21°C and 60 min at 42°C,followed by 5 min at 95°C and 1 min at 98°C for inactivation.The template cDNA obtained was analyzed with PCR as detailedin the previous paragraph.
Induction of Nephritis
Glomerulonephritis was induced in female C57BL/6 mice with asingle injection of 200 µl of sheep anti-mouse GBM seruminto the tail vein, as described previously (16). Age-matchedmice were used as controls. After 6 to 24 h, mice were anesthetizedby intraperitoneal administration of sodium pentobarbital, urinesamples were collected, and kidneys were removed for morphologicanalyses and assays for both calpain and myeloperoxidase (MPO)activities. Urine samples were centrifuged immediately at 1800rpm during 10 min at 4°C, and supernatants were frozen at–20°C. Kidneys were snap-frozen or fixed in 4% paraformaldehydeand processed for paraffin embedding.
Assessment of Albuminuria
Urine albumin concentration was determined by an ELISA assay(Albuwell; Exocell, Philadelphia, PA), and albuminuria was reportedto creatininuria as measured by a colorimetric method.
Histologic and Immunofluorescence Preparations
Renal fragments that were embedded in paraffin were cut into3-µm sections and stained with Masson’s trichromefor histologic analysis. Calpastatin expression was assessedusing a polyclonal primary antibody (Affinity BioReagents, Golden,CO; 1:200). Samples were revealed with Envision System (Dako,Glostrup, Denmark) and counterstained with hematoxylin. No primaryantibody was used for negative control. Fibrin deposition inglomeruli was counted on a minimum of 50 glomeruli by two independentexaminers who were unaware of the genotypes of the mice. Fibrindeposition also was detected by immunofluorescence on cryosectionsthat were fixed for 10 min in 4% paraformaldehyde and stainedfor 30 min in FITC-conjugated goat anti-mouse fibrin (NordiskImmunol, Paris la Défense, France; 1:50). Polymorphonuclearneutrophils that infiltrated glomeruli were stained with hematoxylinand naphthol AS-D chloroacetate esterase (Sigma Chemical Co.,St. Louis, MO) to be counted.
To analyze nephrin expression, 4-µm-thick cryostat sectionswere fixed in 3.5% paraformaldehyde for 15 min and washed inPBS. The sections were incubated with an anti-nephrin antibodythat specifically recognizes the extracellular fibronectin domainof nephrin (GP-N1; Progen Biotechnic, Heidelberg, Germany; 1:50)for 1 h at room temperature, washed in PBS, and incubated withthe appropriate FITC-conjugated secondary antibody (Sigma) for30 min at room temperature. The number of glomeruli that wereavailable on each section for analysis of nephrin expressionranged between five and 10.
To analyze nephrin expression on cultured podocytes, immunofluorescenceexperiments were performed as described previously (17). Briefly,podocytes were plated in eight-well Permanox slide at densityof 40,000 cells per well in DMEM plus 10% FCS. For determinationof whether nephrin expression was modified by exogenous µ-calpain,cells were incubated with variable concentrations of µ-calpainor vehicle alone (0.1% BSA) for 30 min before fixation with3.5% paraformaldehyde that contained 2% sucrose for 15 min atroom temperature. Nonpermeabilized cells then were blocked for30 min with 3% BSA and stained for 60 min at 37°C with anti-nephrinantibody (GP-N1, 1:50) before incubation for 30 min at roomtemperature with the appropriate FITC-conjugated secondary antibody(Sigma). The slides then were mounted with Vectashield mountingmedium (Vector Laboratories, Burlingame, CA) and examined. Controlexperiments included incubation of sections or cells with nonimmuneisotypic control antibodies or the omission of primary antibodiesfollowed by the appropriate labeled secondary antibodies.
Nephrin expression was analyzed semiquantitatively by measuringfluorescence intensity by digital image analysis (Windows MicroImage,version 3.4; CASTI Imaging, Venice, Italy) on images that wereobtained using a low-light video camera (Leica DC100, RueilMalmaison, France) with a 180-µm diameter field. The resultsare expressed as relative fluorescence intensity on a scalefrom 0 (fluorescence of background of tissue) to 255 (fluorescenceof standard filter). For each experimental point on culturedpodocytes, a minimum of five microscopic fields were examined.
Calpain Activity Assay
For measuring calpain activity in urine and plasma, 10 µlof either fluid sample was diluted one sixth in KRB solution(pH 7.4) supplemented with CaCl2 (2 mM final concentration)in wells of a 96-well plate. These samples were exposed at 37°Cto the substrate N-succinyl-Leu-Tyr-AMC 50 µM togetherwith or without the calpain inhibitor calpeptin (100 µM;Calbiochem, VWR International, Fontenay sous Bois, France).After a 90-min incubation period, fluorescence was detectedat 360 nm excitation and 460 nm emission, using the FLX800 spectrofluorometer(Bio-Tek Instruments, Winoski, UT). Calpain activity was determinedas the difference between fluorescences measured without andwith calpeptin and expressed as µM AMC using a standardcurve (0 to 25 µM) constructed for each assay.
For measuring calpain activity in cell supernatant, cells wereplated in 12-well tissue culture dishes in appropriate medium.After the indicated culture period, the medium was replacedwith KRB solution (pH 7.4) that contained 2 mM CaCl2 and 20µM N-succinyl-Leu-Leu-Val-Tyr-AMC together with or withoutcalpeptin 100 µM. Calpain activity was determined in supernatantsas indicated in the previous paragraph.
MPO Assay
Snap-frozen kidney samples were thawed, added to ice-cold 50mM PBS (pH 6.0) supplemented with 0.5% hexadecyltrimethylammoniumbromide up to concentrations of 0.1 g renal tissue/ml, and homogenizedwith 20 strokes in a glass homogenizer. The lysates then werefreeze-thawed three times and centrifuged at 20,000 x g for1 h at 4°C. Supernatants were assayed for MPO, as describedpreviously (18). Results were expressed as OD/min.
NF-B Activation Assay
Nuclear proteins were extracted from fresh kidney samples, andthe amounts of activated NF-B p65 subunit that was containedin these proteins were measured with commercial kits (NuclearExtract Kit and TransAM; Active Motif, Rixensart, Belgium),according to the manufacturer’s instructions.
Western Blotting
Half-kidneys were homogenized in 400 µl of radioimmunoprecipitationassay buffer, as described previously (19). Protease inhibitorcocktail (1 µg/ml; Sigma) was added to the radioimmunoprecipitationassay buffer just before use. After homogenization, the lysatewas centrifuged at 1000 x g for 1 h, and the supernatant wasfrozen at –80°C. Protein concentration in supernatantwas measured using the Bradford method. Twenty-five microgramsof protein was separated by electrophoresis on a Bis-Tris gel4 to 12% (Invitrogen). After transfer of the proteins for 3h onto nitrocellulose membrane, the membrane was incubated for2 h at room temperature with anti-spectrin mAb (1:5000; ChemiconInternational, Temecula, CA). Thereafter, the membrane was incubatedfor 2 h at room temperature with a secondary anti-mouse IgGantibody (1:4000) and developed with the ECL detection reagent(Amersham Pharmacia Biotech). Comparative densitometry was performedon the 145/150-kD spectrin breakdown products (BDP).
The same protocol was used for assessing m-calpain expressionin urine (2 µl) and cell supernatant (10 µl). Firstand secondary antibodies used were goat anti–m-calpainpolyclonal antibody (1:3000; Santa Cruz Biotechnology) and anti-goatIgG antibody (1:10,000), respectively.
Statistical Analyses
Values are expressed as mean ± SEM. Comparisons betweentwo groups of values were made with the t test. Multiple groupcomparisons were performed using the ANOVA with Bonferroni posttest. P < 0.05 was considered statistically significant.
Calpain Activity Was Increased in Kidney Cortex and Released in Urine at the Heterologous Phase of Anti-GBM Nephritis in Wild-Type Mice
To assess initially the role of calpains in renal inflammation,we induced an anti-GBM nephritis in C57BL/6 mice. Kidneys wereremoved 24 h after injection of anti-GBM antiserum, and calpainactivity was determined in the renal cortex by measurement ofthe accumulation of 145/150-kD spectrin BDP that is known tobe generated specifically by calpains. The renal level of BDPwas increased significantly in nephritic mice (Figure 1). Theupregulation was evident from 0.5 h after injection and sustainedto 48 h (i.e., during all of the heterologous phase of nephritis;data not shown). Because several groups showed that calpainsmay be released from cells into the extracellular environmentand therefore may have an extracellular role, we also determinedthe appearance of calpain activity in urine by measuring thecalpain-specific cleavage of fluorescence AMC substrate. Inmost cases, urine calpain activity was undetectable in saline-treatedmice, whereas it was markedly increased in nephritic mice (Figure 2A).This apparent release of calpain in urine of nephritic micewas confirmed by Western blot analysis using an antibody thatwas directed against m-calpain (Figure 2B).
Figure 1. Renal activity of calpains in anti–glomerular basement membrane (anti-GBM) nephritis. (A) Wild-type (WT) mice and calpastatin-transgenic mice (TG) received an injection of either saline (cont) or anti-GBM antiserum (neph), and kidneys were removed at 24 h. Calpain activity was determined in the renal cortex by measurement of the accumulation of 145/150-kD spectrin breakdown products. One representative Western blot is shown. (B) Densitometry analysis of the results. The number of mice per group is noted in parentheses at the bottom of each column. *P < 0.05 versus WT cont; #P < 0.05 versus WT neph.
Figure 2. Urine calpain activity. Urine samples were collected from C57BL/6 mice that received an injection of anti-GBM antiserum and from Nphs 2–/– mice. (A) Urine calpain activity was determined by measurement of the calpain-specific cleavage of fluorescence AMC. The number of mice per group is noted in parentheses at the bottom of each column. (B) Urine samples were collected at 6 and 24 h and immunoblotted for calpain protein by means of an antibody that recognizes m-calpain. One representative experiment of three is shown.
Urine Calpain Activity Originated from Multiple Potential Sources
There are at least three different ways in which the appearanceof calpain in the urine might happen: The release of calpainfrom inflammatory cells that infiltrate the glomerulus, theabnormal transglomerular passage of plasma proteins (includingcalpain) as a result of increased permeability of glomerularcapillary wall, and the secretion of calpain by tubular epithelialcells that are exposed to proteinuria. First, to test the possibilitythat urinary calpain originates from inflammatory cells, wedetermined in vitro the appearance of calpain activity in themicroenvironment of polymorphonuclear neutrophils (105/ml) thatwere isolated from the blood of C57BL/6 mice. After 1 h of incubationwithout and with 10 ng/ml TNF-, extracellular calpain activityreached 0.10 ± 0.06 and 4.70 ± 1.22 µM AMC,respectively (n = 4). Second, to address the role of alterationsin the glomerular filtration barrier, we analyzed calpain activityin the urine of podocin-deficient (Nphs 2–/–) mice,which develop massive proteinuria in the absence of glomerularinflammation. Elevations in urine calpain activity were similarin those mice and in mice with anti-GBM nephritis, suggestingthat calpain appearance in the urine of mice with anti-GBM nephritiscould result from the abnormal transglomerular passage of plasmaproteins, irrespective of inflammatory process (Figure 2A).Accordingly, calpain activity was detectable in plasma of bothcontrol and nephritic mice (4.41 ± 0.90 and 4.78 ±0.52 µM AMC, respectively; n = 7 to 14). Third, we analyzedin vitro the leakage of calpain activity from tubular epithelialcells. We found that HK-2 cells that were exposed to albuminreleased calpain activity in a dose-dependent manner, as determinedby measuring the calpain-specific cleavage of fluorescence AMCsubstrate (Figure 3). These data suggested that urine calpainalso might result, at least partially, from tubular secretion.
Figure 3. Identification of calpain activity in the extracellular medium of HK-2 cells. HK-2 cells were exposed for 6 h to the indicated concentrations of albumin. Extracellular calpain activity was determined by measurement of the calpain-specific cleavage of fluorescence AMC substrate in cell supernatants. *P < 0.05 versus control without albumin (n = 5).
Calpastatin-Transgenic Mice Developed Less Severe Glomerular Inflammation Than Wild-Type Mice
To determine the exact role of both intracellular and externalizedcalpain activities in the development of renal inflammation,we induced an anti-GBM nephritis in mice that were transgenicfor calpastatin and compared them with wild-type mice from thesame genetic background. The presence and the expression ofthe transgene were identified in homozygous calpastatin-transgenicmice by PCR and RT-PCR analysis, respectively (Figure 4A). Thesemice showed normal development, fertility, morphology, and lifespan as compared with wild-type mice. To localize the transgeneproduct in the kidney, we compared the expression of calpastatinin wild-type and calpastatin-transgenic mice. As assessed byimmunohistochemistry, only a few tubules showed calpastatinstaining in wild-type mice (Figure 4B). In calpastatin-transgenicmice, the intensity of the staining and the number of calpastatin-positivetubules increased markedly. By contrast, calpastatin expressionwas barely detectable in glomeruli, in both wild-type and calpastatin-transgenicmice.
Figure 4. Identification and characterization of TG mice. (A) The presence and the expression of the transgene were identified in TG mice by PCR (left) and reverse transcriptase–PCR (RT-PCR) analysis (right), respectively. DNA and RNA were isolated from mouse tails that were biopsied after 3 wk of age. RT-PCR was performed with (RT+) or without (RT–) reverse transcriptase to control for DNA contamination. The 518-bp product corresponds to rabbit calpastatin gene. (B) The renal expression and localization of calpastatin were compared in WT and TG mice by immunohistochemistry. Magnification, x200.
Whereas calpastatin overexpression did not affect calpain activityin control mice, it prevented completely calpain activationin the kidney of mice with anti-GBM nephritis (Figure 1). Inthose mice, calpain release in the urine also was reduced, albeitnot significantly (from 0.159 ± 0.037 to 0.090 ±0.012 µM AMC/mM creatinine; n = 7 and 5, respectively).The decreased activity of both intracellular and externalizedcalpains was accompanied by a lessening in the severity of glomerularinjury. Despite an equivalent deposition of heterologous sheepantibody (data not shown), glomerular fibrin deposition wasmarkedly attenuated in calpastatin-transgenic mice, as reflectedby the decrease in fibrin immunoreactivity (Figure 5). The numberof polymorphonuclear neutrophils that infiltrated glomerulidid not change (16.6 ± 5.5 versus 16.9 ± 2.8 neutrophils/50glomeruli in section; n = 3 for each group), but their activitywas markedly reduced, as reflected by the decrease in MPO activity(Figure 5). To further characterize the mechanisms whereby calpastatinoverexpression would limit inflammatory process, we quantifiedNF-B activity in nuclear extracts that were obtained from kidneycortex. Six hours after injection of anti-GBM, nuclear NF-Bactivity increased markedly in wild-type mice while increasingweakly in calpastatin-transgenic mice (Figure 6).
Figure 5. Glomerular fibrin deposition and neutrophil activity are decreased in nephritic TG mice as compared with nephritic WT mice. WT mice and TG mice received an injection of anti-GBM antiserum. (A and B) Glomerular fibrin deposition was assessed by fluorescence (top) and light (bottom) microscopy 24 h after injection of anti-GBM antiserum. *P < 0.05 versus WT mice (n = 8). (C) Myeloperoxidase activity of neutrophils that infiltrated glomeruli was measured 6 h after injection of anti-GBM antiserum. *P < 0.05 versus WT cont; #P < 0.05 versus WT neph (n = 5).
Figure 6. Renal NF-B activity is decreased in nephritic TG mice as compared with nephritic WT mice. WT mice and TG mice received an injection of anti-GBM antiserum. Kidney cortex samples were collected at 6 h and nuclear NF-B activity was measured. *P < 0.05 versus WT cont; #P < 0.05 versus WT neph.
Calpastatin-Transgenic Mice Developed Less Severe Albuminuria Than Wild-Type Mice
Development of albuminuria, which peaked at 24 h (heterologousphase) and again at 15 d (autologous phase), was reduced significantlyin calpastatin-transgenic mice at these two phases (Figure 7).Because nephrin is a critical component of the barrier to proteinpermeation, we analyzed nephrin expression. In wild-type controlmice, nephrin exhibited a glomerular epithelial pattern witha punctate/linear distribution. In glomeruli of wild-type micewith an anti-GBM nephritis, a more granular pattern or a lossof staining of nephrin was observed 24h after induction of thedisease (Figure 8). Anti-GBM–induced loss of nephrin expressionwas limited significantly in calpastatin-transgenic mice. Therefore,we tested the hypothesis that urinary calpain activity wouldparticipate in the development of proteinuria by altering nephrinexpression. To this aim, we analyzed the effect of externalizedcalpains on nephrin expression by podocytes in vitro. When testedby indirect immunofluorescence, antibody that was specific forthe extracellular domain of nephrin bound to both human podocytesin primary culture and human immortalized podocytes, suggestinga surface expression of nephrin (data not shown). Exposure ofthose cells to calpain promoted the disappearance of nephrinfrom the surface, as indicated by a significant decrease inimmunofluorescence staining (Figure 9A). Similarly, the appearanceof calpain activity in the culture medium of podocytes thatwere exposed to TNF- for 1 h (1.76 ± 0.20 µM AMC;n = 3) was concomitant with a significant decrease in nephrinexpression (Figure 9B). Under these conditions, blocking extracellularcalpain activity by adding the non–cell-permeant calpastatinprevented completely nephrin disappearance from the cell surface,confirming a role for extracellular calpain activity in thisprocess.
Figure 7. Albuminuria is decreased in nephritic TG mice as compared with nephritic WT mice. WT mice () and TG mice () received an injection of anti-GBM antiserum. Urine albumin concentration was determined by ELISA. *P < 0.05 versus WT mice (n = 5).
Figure 8. Nephrin expression in vivo. (A) Immunofluorescence staining for nephrin in glomeruli of control and nephritic mice 24 h after injection of anti-GBM antiserum. (B) Semiquantitative analysis of nephrin expression in glomeruli of nephritic WT mice () as compared with nephritic TG mice (). *P < 0.05 versus WT mice (n = 3). Magnification, x400.
Figure 9. Effect of extracellular calpain on nephrin expression in vitro. (A) Human immortalized podocytes were exposed for 30 min to µ-calpain before nephrin expression was determined by immunofluorescence technique. *P < 0.05 versus control cells (n = 3). (B) Human podocytes were incubated first with or without calpastatin (0.5 µM) for 15 min and then with or without TNF- (10 ng/ml) for 1 h, before semiquantitative analysis of nephrin expression was performed. *P < 0.05 versus control cells (n = 3).
Calpains are intracellular proteases that potentially are involvedin the pathophysiology of inflammation. According to in vitroexperiments, they are responsible primarily for the activationof NF-B, a transcription factor with a pivotal role in inflammation(6). They play a key role in inflammatory cell adhesion andmigration, proinflammatory mediator release, and anti-inflammatoryhormone resistance as well (7–9). In addition, becausethey are precursors of chemotactic factors, their release inthe microenvironment of inflammatory cells would amplify neutrophilrecruitment (11). Studies of a variety of experimental modelsof inflammation have confirmed the relevance of these processesin vivo. However, attempts to intervene in calpain activationin these models have been made by using synthetic peptidic andnonpeptidic calpain inhibitors that have problems in terms ofspecificity and stability. To overcome these drawbacks and togain new insights into the pathophysiologic roles of calpainsin glomerular inflammation, we induced an anti-GBM nephritisin mice that were transgenic for calpastatin, the specific endogenousinhibitor of calpain, and we compared them with wild-type micefrom the same genetic background.
Whereas RT-PCR studies confirmed calpastatin transgene expressionin homozygous calpastatin-transgenic mice, there was no evidenceof calpain inactivation in the kidney cortex of those mice undernormal conditions, as determined by measuring the accumulationof calpain-specific spectrin BDP. Recently, Takano et al. (20)also observed that basal calpain activity remained unchangedin the brain of calpastatin-transgenic mice as compared withwild-type controls. Altogether, these results support the ideathat calpastatin levels are physiologically sufficient to controlfully calpain activity under normal conditions and/or that calpastatincontrols calpain activity only under pathologic conditions.In line with the latter hypothesis, these studies demonstratedthat renal injury in the early heterologous phase of anti-GBMnephritis was associated with an increase in the renal activityof calpain in wild-type mice but not in calpastatin-transgenicmice. The only published reports of enhanced calpain activityin kidney disease were so far limited to models of ischemia-reperfusion(19).
Our study was the first demonstration that calpain secretionin urine paralleled calpain activation in renal cortex duringthe early course of anti-GBM nephritis. The peak expressionin urine occurred at 24 h, when albuminuria was the most abundant,suggesting that lesions of the glomerular filtration barrierplayed a major role. Consistent with this hypothesis, we detectedhigh amounts of calpain activity in the urine of Nphs 2–/–mice, which develop massive proteinuria in the absence of glomerularinflammation (21). These events suggest that calpain is presentin plasma and is filtered through the injured glomerular filtrationbarrier. Accordingly, calpain activity was evidenced in plasmaof mice with anti-GBM nephritis, as previously shown in plasmaof rats with CCl4-induced hepatitis (22). Another possible mechanismof calpain appearance in urine of anti-GBM nephritic mice isits secretion by tubular epithelial cells. Indeed, as otherintracellular enzymes, calpains may leak out from injured anddying cells such as hepatocytes that are exposed to toxic chemicals(22) or tubular epithelial cells that are submitted to hypoxia(23). Our finding that epithelial cells of proximal tubule (HK-2cells) that were exposed to albumin released calpain in theextracellular milieu supports this hypothesis. It is interestingthat exposure of renal proximal tubular cells to albumin wasshown recently to induce endoplasmic reticulum stress and calpainactivation, in turn responsible for caspase activation and apoptosis(24). It is possible, therefore, that calpain activation andrelease are linked in tubular epithelial cells. Although themechanisms of calpain release from HK-2 cells that were exposedto albumin were not examined in our study, the observation thatcalpain release from osteoblasts and parathyroid cells is dueto the shedding of membrane vesicles (25,26) suggests possiblepathways of secretion to explore.
The results reported here showed that calpain inactivation incalpastatin-transgenic mice was effective in reducing the severityof glomerular inflammation. The marked reduction in the incidenceof glomerular capillary thrombi may be due to interference withthe TNF-–induced expression of procoagulant activity byendothelial and mesangial cells. Indeed, tissue factor expressionis responsible for glomerular fibrin deposition in experimentalmodels of anti-GBM nephritis (27), and TNF- plays a criticalrole in tissue factor production by glomerular cells (28). AsNF-B is the main factor involved in the transcriptional expressionof TNF-, calpain inactivation in calpastatin-transgenic micewould prevent NF-B activation and thereby limit TNF-–dependentexpression of tissue factor. The progressive activation of NF-Bthat is observed at the onset of anti-GBM nephritis (29) andits marked limitation in calpastatin-transgenic mice supportthis possibility.
These studies demonstrated also a decrease in glomerular injuryand albuminuria in nephritic calpastatin-transgenic mice ascompared with nephritic wild-type mice. This protection wasassociated with the persistence of nephrin expression, suggestinga role for calpain in nephrin redistribution. There are a numberof different ways in which calpain activity might affect nephrinexpression. First, calpain is known to cleave kinases and phosphatasesas well as many of the cytoskeletal proteins that are involvedin cytoskeletal-plasma membrane interactions (3). Therefore,it is conceivable that intracellular calpain activity promotesa slit diaphragm disruption through loss of the nephrin–actinconnection, either directly or indirectly through alterationsin the phosphorylation state of nephrin (30). Second, TNF- hasbeen shown to induce the shedding of nephrin from podocytesin culture (17). This suggests that calpain activity might amplifyTNF-–dependent loss of nephrin expression indirectly throughNF-B–dependent TNF- gene transcription. Nevertheless,these two mechanisms are insufficient to explain our in vitroresults, because nephrin expression by podocytes was affectedby extracellular rather than intracellular calpain. Therefore,the most attractive explanation is that extracellular calpaincleaves directly the extracellular domain of nephrin and therebypromotes its shedding.
These results are in agreement with our hypothesis that tissuecalpain activity plays a critical role in glomerular inflammationand injury in experimental glomerulonephritis and that its inhibitionmay have therapeutic benefits. They also are the first to showthat urinary calpain activity participates in nephrin redistribution,thereby strongly indicating its role as a marker and also amediator of renal injury.
Acknowledgments
This work was supported by the Institut National de la Santéet de la Recherche Médicale and by the Facultéde Médecine Saint-Antoine. Additional support was providedby grants from the Association pour la Recherche sur le Cancer(9946), the Ligue Nationale contre le Cancer (Comitéde Paris), and the Baxter Extramural Grant Program. During partof this work, Julie Peltier was supported by a grant from theSociété de Néphrologie. Sophie Doublieris recipient of a Research Fellowship from the Fondazione Internazionaledi Ricerca in Medicina Sperimentale (Turin, Italy).
We are indebted to Dr. Jérôme Rossert and CatherineTerraz for help with the transgenic mouse constructs and toDr. Corinne Antignac for providing urine samples from Nphs2–/–mice.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Schrijver G, Schalkwijk J, Robben JCM, Assmann KJM, Koene RAP: Antiglomerular basement membrane nephritis in beige mice. Deficiency of leukocytic neutral proteinases prevents the induction of albuminuria in the heterologous phase.
J Exp Med 169
: 1435
–1448, 1989[Abstract/Free Full Text]
Goll DE, Thompson VF, Li H, Wei W, Cong J: The calpain system.
Physiol Rev 83
: 731
–801, 2003[Abstract/Free Full Text]
Zatz M, Starling A: Calpains and disease.
N Engl J Med 352
: 2413
–2423, 2005[Free Full Text]
Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z, Davies PL: A Ca2+ switch aligns the active site of calpain.
Cell 108
: 649
–660, 2002[CrossRef][Medline]
Shumway SD, Maki M, Miyamoto S: The PEST domain of IkappaBalpha is necessary and sufficient for in vitro degradation by mu-calpain.
J Biol Chem 274
: 30874
–30881, 1999[Abstract/Free Full Text]
Lokuta MA, Nuzzi PA, Huttenlocher A: Calpain regulates neutrophil chemotaxis.
Proc Natl Acad Sci U S A 100
: 4006
–4011, 2003[Abstract/Free Full Text]
Kavita U, Mizel SB: Differential sensitivity of interleukin-1 alpha and beta precursor proteins to cleavage by calpain, a calcium-dependent protease.
J Biol Chem 270
: 27758
–27765, 1995[Abstract/Free Full Text]
Bellocq A, Doublier S, Suberville S, Perez J, Escoubet B, Fouqueray B, Rodriguez Puyol D, Baud L: Somatostatin increases glucocorticoid binding and signaling in macrophages by blocking the calpain-specific cleavage of Hsp 90.
J Biol Chem 274
: 36891
–36896, 1999[Abstract/Free Full Text]
Menard H-A, El-Amine M: The calpain-calpastatin system in rheumatoid arthritis.
Immunol Today 17
: 545
–547, 1996[CrossRef][Medline]
Kunimatsu M, Ma XJ, Nishimura J, Baba S, Hamada Y, Shioiri T, Sasaki M: Neutrophil chemotactic activity of N-terminal peptides from the calpain small subunit.
Biochem Biophys Res Commun 169
: 1242
–4247, 1990[CrossRef][Medline]
Cuzzocrea S, McDonald MC, Mazzon E, Siriwardena D, Serraino I, Dugo L, Britti D, Mazzullo G, Caputi AP, Thiemermann C: Calpain inhibitor I reduces the development of acute and chronic inflammation.
Am J Pathol 157
: 2065
–2079, 2000[Abstract/Free Full Text]
Chatterjee PK, Todorovic Z, Sivarajah A, Mota-Filipe H, Brown PAJ, Stewart KN, Mazzon E, Cuzzocrea S, Thiemermann C: Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury.
Biochem Pharmacol 69
: 1121
–1131, 2005[CrossRef][Medline]
Arthur JSC, Elce JS, Hegadorn C, Williams K, Greer PA: Disruption of the murine calpain small subunit gene, Capn4: Calpain is essential for embryonic development but not for cell growth and division.
Mol Cell Biol 20
: 4474
–4481, 2000[Abstract/Free Full Text]
Conaldi PG, Biancone L, Bottelli A, De Martino A, Camussi G, Toniolo A: Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.
J Virol 71
: 9180
–9187, 1997[Abstract]
Lelongt B, Bengatta S, Delauche M, Lund LR, Werb Z, Ronco PM: Matrix metalloproteinase 9 protects mice from anti-glomerular basement membrane nephritis through its fibrinolytic activity.
J Exp Med 193
: 793
–802, 2001[Abstract/Free Full Text]
Doublier S, Ruotsalainen V, Salvidio G, Lupia E, Biancone L, Conaldi PG, Reponen P, Tryggvason K, Camussi G: Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.
Am J Pathol 158
: 1723
–1731, 2001[Abstract/Free Full Text]
Shi Y, Melnikov VY, Schrier RW, Edelstein CL: Downregulation of the calpain inhibitor protein calpastatin by caspases during renal ischemia-reperfusion.
Am J Physiol Renal Physiol 279
: F509
–F517, 2000[Abstract/Free Full Text]
Takano J, Tomioka M, Tsubuki S, Higuchi M, Iwata N, Itohara S, Maki M, Saido TC: Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains. Evidence from calpastatin mutant mice.
J Biol Chem 280
: 16175
–16184, 2005[Abstract/Free Full Text]
Roselli S, Heidet L, Sich M, Henger A, Kretzler M, Gubler M-C, Antignac C: Early glomerular filtration defect and severe renal disease in podocin-deficient mice.
Mol Cell Biol 24
: 550
–560, 2004[Abstract/Free Full Text]
Limaye PB, Apte UM, Shankar K, Bucci TJ, Warbritton A, Mehendale HM: Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants.
Toxicol Appl Pharmacol 191
: 211
–226, 2003[CrossRef][Medline]
Liu X, Rainey JJ, Harriman JF, Schnellmann RG: Calpains mediate acute renal cell death: Role of autolysis and translocation.
Am J Physiol Renal Physiol 281
: F728
–F738, 2001[Abstract/Free Full Text]
Ohse T, Tanaka T, Kojima I, Inagi R, Miyata T, Ingelfinger JR, Fujita T, Nangaku M: Chronic proteinuria induces endoplasmic reticulum (ER) stress on proximal tubular cells and accelerates ER-associated apoptosis via calpain and caspase-12 [Abstract].
J Am Soc Nephrol 16
: 220A
–226, 2005
Nishihara H, Nakagawa Y, Ishikawa H, Ohba M, Shimizu K, Nakamura T: Matrix vesicles and media vesicles as nonclassical pathways for the secretion of m-calpain from MC3T3–E1 cells.
Biochem Biophys Res Commun 285
: 845
–853, 2001[CrossRef][Medline]
Kifor O, Kifor I, Moore FD, Butters RR, Brown EM: m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR.
J Biol Chem 278
: 31167
–31176, 2003[Abstract/Free Full Text]
Erlich JH, Holdsworth SR, Tipping PG: Tissue factor initiates glomerular fibrin deposition and promotes major histocompatibility complex class II expression in crescentic glomerulonephritis.
Am J Pathol 150
: 873
–880, 1997[Abstract]
Wiggins RC, Njoku N, Sedor JR: Tissue factor production by cultured rat mesangial cells. Stimulation by TNF alpha and lipopolysaccharide.
Kidney Int 37
: 1281
–1285, 1990[Medline]
Kim JH, Ha IS, Hwang C-I, Lee Y-J, Kim J, Yang S-H, Kim YS, Cao YA, Choi S, Park W-Y: Gene expression profiling of anti-GBM glomerulonephritis model: The role of NF-kappaB in immune complex kidney disease.
Kidney Int 66
: 1826
–1827, 2004[CrossRef][Medline]
Saran AM, Yuan H, Takeuchi E, McLaughlin M, Salant DJ: Complement mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.
Kidney Int 64
: 2072
–2078, 2003[CrossRef][Medline]
This article has been cited by other articles:
T. Fujimura, S.-i. Yamagishi, S. Ueda, K. Fukami, R. Shibata, Y. Matsumoto, Y. Kaida, A. Hayashida, K. Koike, T. Matsui, et al. Administration of pigment epithelium-derived factor (PEDF) reduces proteinuria by suppressing decreased nephrin and increased VEGF expression in the glomeruli of adriamycin-injected rats
Nephrol. Dial. Transplant.,
November 28, 2008;
(2008)
gfn659v1.
[Abstract][Full Text][PDF]
E. Letavernier, J. Perez, A. Bellocq, L. Mesnard, A. de Castro Keller, J.-P. Haymann, and L. Baud Targeting the Calpain/Calpastatin System as a New Strategy to Prevent Cardiovascular Remodeling in Angiotensin II-Induced Hypertension
Circ. Res.,
March 28, 2008;
102(6):
720 - 728.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
B activation, suggesting that calpain may participate in inflammatory lesions through NF-
from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury. 
) and TG mice (
) received an injection of anti-GBM antiserum. Urine albumin concentration was determined by ELISA. *P < 0.05 versus WT mice (n = 5).
