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c-mip Down-Regulates NF-κB Activity and Promotes Apoptosis in Podocytes

Open ArchivePublished:April 16, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.02.008
      The mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS.
      Idiopathic nephrotic syndrome (INS) defines several entities, including minimal change nephrotic syndrome (MCNS) and focal and segmental glomerulosclerosis (FSGS), which are considered to be prototypic podocyte diseases.
      • Mathieson P.W.
      Minimal change nephropathy and focal segmental glomerulosclerosis.
      Podocytes are terminally differentiated cells that line the outer aspect of the glomerular basement membrane and constitute the ultimate barrier to urinary protein loss by the formation and maintenance of the podocyte foot processes and the interposed slit diaphragm.
      • Tryggvason K.
      • Patrakka J.
      • Wartiovaara J.
      Hereditary proteinuria syndromes and mechanisms of proteinuria.
      Podocyte diseases might result from genetic defects of proteins playing a key structural and/or regulatory role in the integrity of the glomerular filtration barrier.
      • Shankland S.J.
      The podocyte's response to injury: role in proteinuria and glomerulosclerosis.
      Regardless of the underlying cause, the early podocyte damages are characterized by ultrastructural alterations of the slit diaphragm. Cellular injuries may progress through a reversible stage to podocyte depletion and glomerulosclerosis, which commonly complicate the outcome of chronic glomerular diseases in INS. In acquired INS, the mechanisms preceding these changes remain to be elucidated.
      The NF-κB family of transcription factors plays a central role in many cellular processes through the regulation of genes involved in immunity, inflammation, cell proliferation, differentiation, and apoptosis. In mammalian cells, the NF-κB family consists of five members, NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB, and cRel.
      • Shih V.F.
      • Tsui R.
      • Caldwell A.
      • Hoffmann A.
      A single NF-kappa B system for both canonical and non-canonical signaling.
      The hallmark of the NF-κB family is the presence in all members of a Rel-homology domain at the NH2-teminus, responsible for DNA binding, dimerization, and association with the I-κB inhibitory proteins.
      • Zheng C.
      • Yin Q.
      • Wu H.
      Structural studies of NF-kappa B signaling.
      Among the NF-κB proteins, only RelA, RelB, and c-Rel have a COOH-terminus transactivation domain, which is lacking in p50 and p52, so that RelA, RelB, and c-Rel homo- or heterodimers function as transcriptional activators, whereas p50 and p52 homodimers function as repressors.
      • Shih V.F.
      • Tsui R.
      • Caldwell A.
      • Hoffmann A.
      A single NF-kappa B system for both canonical and non-canonical signaling.
      Although in most cases, Rel proteins act as transcriptional activators, they may repress some target genes.
      • Fu T.
      • Li P.
      • Wang H.
      • He Y.
      • Luo D.
      • Zhang A.
      • Tong W.
      • Zhang L.
      • Liu B.
      • Hu C.
      c-Rel is a transcriptional repressor of EPHB2 in colorectal cancer.
      • Ashburner B.P.
      • Westerheide S.D.
      • Baldwin Jr., A.S.
      The p65 (RelA) subunit of NF-kappa B interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression.
      Because of its implication in many biological processes and the adverse consequences of its dysregulation, the activity of NF-κB is tightly regulated at multiple translational and posttranslational levels. The most common regulatory mechanism relies on the actions of the inhibitory I-κB proteins.
      • Ferreiro D.U.
      • Komives E.A.
      Molecular mechanisms of system control of NF-kappa B signaling by I-kappa B alpha.
      In resting cells, I-κB protein binds to the nuclear localization site of the Rel-homology domain, preventing the nuclear translocation of NF-κB protein and thereby its transcriptional activity. On activation, I-κB protein is phosphorylated, which allows its ubiquitination and subsequent degradation by proteasome. We recently shown that c-mip interacts with RelA and prevents its dissociation from the NFκB/IκBα complexes, resulting in the inhibition of nuclear translocation of NFκB and stabilization of IκBα.
      • Kamal M.
      • Valanciute A.
      • Dahan K.
      • Ory V.
      • Pawlak A.
      • Lang P.
      • Guellaen G.
      • Sahali D.
      C-mip interacts physically with RelA and inhibits nuclear factor kappa B activity.
      In human and experimental glomerular diseases, several mediators regulated by NF-κB such as cytokines and adhesion molecules have been reported to play pathogenic roles in inflammatory and proliferative glomerular diseases.
      • Mudge S.J.
      • Paizis K.
      • Auwardt R.B.
      • Thomas R.J.
      • Power D.A.
      Activation of nuclear factor-kappa B by podocytes in the autologous phase of passive Heymann nephritis.
      In nonproliferative glomerular diseases characterized by heavy proteinuria, such as minimal change disease and membranous nephropathy, it has been suggested that the excess of proteins in the tubules and their subsequent degradation induce tubular expression of many proinflammatory genes through NF-κB activation.
      • Remuzzi G.
      • Ruggenenti P.
      • Benigni A.
      Understanding the nature of renal disease progression.
      To our knowledge, the mechanisms of NF-κB regulation in the podocytes are little known. We recently reported that c-mip is overproduced in the podocytes during glomerular damages and interferes with signaling pathways playing a key role in podocyte function. Whether c-mip influences NF-κB activity has not been investigated. We report here that c-mip represses in vitro and in vivo NF-κB activity by down-regulating the expression of RelA. This effect was only observed in podocytes, whereas tubular cells displayed a contrasting increase of NF-κB activity. Moreover, c-mip increases the levels of Bax and enhances caspase-3 activity, but it reduces the Bcl-2 level, suggesting that c-mip exerts a proapoptotic function.

      Materials and Methods

       Patients

      The diagnosis of kidney disease was performed by renal biopsy, which was performed before the start of treatment. All patients had proteinuria above 3 g in 24 hours and severe hypoalbuminemia at the time of blood sampling. MCNS and FSGS were clinically classified as idiopathic in all cases.
      Control for c-mip screening includes adult patients with glomerular diseases (IgA, lupus nephritis) who exhibited a nephrotic syndrome. Normal renal samples were supplied by the hospital tissue bank (Platform of Biological Resources, Henri Mondor Hospital) from patients undergoing nephrectomy for polar kidney tumor.

       Plasmid Constructs, Reverse Transcription, and Quantitative PCR

      The expression plasmids for c-mip and RelA have been previously described.
      • Valanciute A.
      • le Gouvello S.
      • Solhonne B.
      • Pawlak A.
      • Grimbert P.
      • Lyonnet L.
      • Hue S.
      • Lang P.
      • Remy P.
      • Salomon R.
      • Bensman A.
      • Guellaen G.
      • Sahali D.
      NF-kappa B p65 antagonizes IL-4 induction by c-maf in minimal change nephrotic syndrome.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      Mouse total RNA was prepared from glomerular fractions isolated by graded sieving, using RNeasy kit (Qiagen, Courtaboeuf, France). Quantitative real-time RT-PCR (RT-qPCR) for RelA was performed using the oligonucleotides sense (5′-TGTTACCATCAGGGCAGATC-3′) and anti-sense (5′-CAGGGTACTCCATCAGCATG-3′). The samples (2 μL of the reverse transcription reaction mixture, corresponding to 20 ng of total RNA) were amplified in a 20-μL reaction mixture containing 0.5 mmol/L of each primer and 1× LightCycler DNA Master SYBR Green buffer (Roche Molecular Biochemical, Mannheim, Germany). RT-qPCR conditions include an initial denaturing step at 95°C for 15 minutes, followed by 40 cycles (denaturing: 95°C, 15 seconds; annealing: 59°C, 30 seconds; extension, 72°C, 35 seconds).

       Generation of c-mip Stable Podocyte Cell Lines

      Conditionally immortalized mouse podocytes have been described elsewhere.
      • Mundel P.
      • Reiser J.
      • Zuniga Mejia Borja A.
      • Pavenstadt H.
      • Davidson G.R.
      • Kriz W.
      • Zeller R.
      Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.
      Before transfection, podocytes were maintained at 60% confluence, under permissive conditions (cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 U/mL γ-INF, at 33°C). The full-length human c-mip was inserted into a pDEST40 plasmid (Invitrogen, Carlsbad, CA). Proliferating podocytes were transfected with c-mip expression plasmid (1 μg/106 cells) using the Amaxa system. Following transfection, cells were maintained in complete medium for 24 hours, then Geneticin (antibiotic G-418 sulfate; Invitrogen) was added at 400 μg/mL (100% lethal to untransfected cells). Stably transfected cells were selected in serial passages using Geneticin, then stored in liquid nitrogen or expanded under permissive conditions in the presence of Geneticin until they were in sufficient number. Podocyte clones were isolated by limiting dilution and stored in liquid nitrogen. For transient transfection, podocytes were transfected with c-mip expression plasmid (pDEST40) alone or cotransfected with empty vector or RelA, using the Nanofectin method according to the instructions provided by the manufacturer (PAA Laboratories, Pasching, Austria). In some experiments, podocytes were incubated with 2 μmol/L lactacystin (Sigma-Aldrich, St. Louis, MO) for 20 hours, and then protein lysates were prepared as previously described.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.

       Caspase-3 Activity Assays

      Caspase-3 activity was monitored in a quantitative assay using a fluorogenic substrate. Podocytes stably transfected with c-mip or expressing empty vector (∼200,000 cells) were grown on 60-mm dishes at 37°C for 96 hours. Floating cells were collected and centrifuged, and the pellet was lysed in 50 μL of lysis buffer containing 50 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 1% Nonidet P-40, 1 mmol/L EDTA (pH 8.0), 1 mmol/L dithiothreitol, 2 μg/mL leupeptin, 2 μg/mL aprotinin. Adherent cells were washed three times with cold PBS and lysed for 10 minutes on ice in 0.2 mL of lysis buffer. The lysates from adherent and floating cells were pooled and centrifuged, and the supernatants were collected. The activity of caspase recognizing the DEVD (Asp-Glu-Val-Asp) motif (DEVDase activity) was measured in 200 μL of assay buffer containing 100 mmol/L HEPES (pH 7.4), 10% sucrose, 10 mmol/L dithiothreitol, 500 μmol/L EDTA, 50 μg of protein, and 20 μmol/L N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (AC-DEVD-AFC) as fluorogenic substrate (Biomol, Tebu, France). After 3 hours at 37°C, the fluorescence of the reaction mixture was monitored every 30 minutes for 7 hours, using a spectrofluorometer (FL600 Fluorescence Microplate Reader; Biotek Instruments, Winooski, VT), with excitation and emission wavelengths of 400 and 530 nm, respectively.

       TUNEL Assays

      Paraffin-embedded kidney tissue sections were dewaxed, rehydrated, and then treated with proteinase K solution [10 μg/mL in 10 mmol/L Tris-HCl (ph7.4)] for 30 minutes at room temperature. The sections were rinsed twice with PBS and the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Mannheim, Germany).was used to label apoptotic cells. The sections were incubated with 50 μL of TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) reaction mixture for 60 minutes at 37°C in a humidified atmosphere in the dark. Terminal transferase was omitted for the negative control. As a positive control, one section was incubated with DNase I [2000 U/mL in 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, and 1 mg/mL BSA] for 10 minutes at room temperature to induce DNA strand breaks, prior to the labeling procedures. The sections were rinsed three times with PBS, and then incubated with 50 μL of Converter-POD on the sample, for 30 minutes at 37°C. The sections were rinsed three times with PBS, and then 50 μL of diaminobenzidine substrate and incubated for several minutes at room temperature. Finally, the sections were rinsed with PBS, mounted under glass coverslips, and analyzed under a light microscope.

       Light Microscopy and Immunohistochemistry Analysis

      For light microscopy, the kidney sections from wild-type and c-mip transgenic (Tg) mice were incubated for 16 hours in Dubosc-Brasil solution, and subsequently dehydrated, paraffin embedded, and stained with periodic acid-Schiff reagent (PAS). For immunohistochemistry study, antigen retrieval was performed by immersing the slides in boiling 0.01 mol/L citrate buffer in a 500-W microwave oven for 15 minutes. The endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 30 minutes. Slides were incubated with the blocking reagents consisting of the avidin–biotin solution for 30 minutes and the normal blocking serum for 20 minutes, and then incubated overnight with a specific polyclonal antibody. After washing with PBS, they were incubated with biotinylated goat anti-rabbit antibody. An avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Reagent; Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine (Sigma Biochemical, St. Louis, MO) as a chromogen were applied for visualization of the immunoreaction. Anti-RelA, anti–c-mip,
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      and anti-cleaved caspase-3 (Asp175) were used at a final concentration of 1:50, 1:200, and 1:30 dilution, respectively. Omission of the primary antibody was considered a negative control.

       Immunofluorescence

      Immunofluorescence studies on kidney tissues were performed with 4-μm-thick cryostat sections fixed in acetone for 10 minutes, air-dried 30 minutes at room temperature, and then kept in PBS for 3 minutes and blocked in 1% BSA-PBS. The sections were incubated with the indicated antibodies for 1 hour at room temperature, washed with PBS, and incubated with fluorescein isothiocyanate–conjugated or red fluorescent dye–conjugated secondary antibodies. For double fluorochrome labeling, the slides were simultaneously incubated with rabbit anti–c-mip antibody and mouse anti-nephrin antibody. After washing with PBS, the slides were simultaneously incubated with fluorescein isothiocyanate–conjugated goat anti-rabbit IgG and cyanine 3–conjugated sheep anti-mouse IgG. Sections were examined by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany) using red and green filters.

       Antibodies and Western Blot Analysis

      The primary antibodies used in this study included anti-RelA, anti-Tie2, anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA), anti-cleaved caspase-3 (Asp 75), anti-Bax, (Cell Signaling Technology, Danvers, MA), anti–Bcl-2 (BD Transduction Laboratories; BD Biosciences, San Jose, CA), monoclonal anti-vimentin (Dako, Glostrup, Denmark), monoclonal anti-GAPDH (Abcam, Cambridge, UK), anti-actin (Sigma-Aldrich), and anti-nephrin (Progen, Heidelberg, Germany). The anti–c-mip polyclonal antibody has been previously described.
      • Audard V.
      • Zhang S.Y.
      • Copie-Bergman C.
      • Rucker-Martin C.
      • Ory V.
      • Candelier M.
      • Baia M.
      • Lang P.
      • Pawlak A.
      • Sahali D.
      Occurrence of minimal change nephrotic syndrome in classical Hodgkin lymphoma is closely related to the induction of c-mip in Hodgkin-Reed Sternberg cells and podocytes.
      Western blot analysis was performed, using classical methods.

       Generation of c-mip Transgenic Mice

      The generation of c-mip transgenic mice has been previously described.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      All experiments involving animals were conducted in accordance with French laws. All of the mice analyzed in this study were hemizygous males [Tg(+)] from the F8 to F12 generations.

       Histological Analysis

      Morphological lesions were studied on 4-mm PAS-stained sections. Five kidney sections by mouse (n = 5 mice for each group) were analyzed. Between 20 and 30 glomeruli per kidney section were randomly selected.

       Statistical Analysis

      Statistical analyses of the data were performed using PRIZM 4 for Macintosh (GraphPad Software, La Jolla, CA). Unpaired or paired Student's t-tests were used. P values of less than 0.05 were considered significant.

      Results

       Expression of c-mip Is Associated With a Down-Regulation of NF-κB in Podocytes of Patients with INS

      Although the role of NF-κB in podocytes is poorly understood, NF-κB likely mediates important functions including regulation of signaling pathways and podocyte survival.
      • Sanz A.B.
      • Sanchez-Nino M.D.
      • Ramos A.M.
      • Moreno J.A.
      • Santamaria B.
      • Ruiz-Ortega M.
      • Egido J.
      • Ortiz A.
      NF-kappa B in renal inflammation.
      To investigate the possible influence of c-mip on NF-κB activity in podocytes of glomerular diseases, we studied the kidney biopsies of patients with INS. As previously reported, c-mip was clearly induced in kidney biopsies of patients with MCNS, but it was scarcely or not detected in normal human kidney.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      The distribution pattern shows that c-mip was not confined to a cytoplasmic compartment, it was also visualized in nuclei (Figure 1A). By contrast, we did not detect c-mip in glomeruli of IgA nephropathy or proliferative lupus nephritis. Immunohistochemistry analysis on the same kidney specimens (5 normal specimens and 12 MCNS) using an anti-RelA antibody showed that RelA was clearly detected in normal podocytes, but it was dramatically altered in the podocytes of MCNS (Figure 1, A and B). Confocal microscopy analysis of normal glomeruli (Figure 2) shows that RelA colocalized partially with nephrin, but not with Tie2, suggesting that its expression is restricted to podocytes and did not overlap with vimentin but mostly colocalized with lamin-B, a marker of nuclear membrane. In some podocytes, RelA was also localized within the nucleus. By contrast, in glomeruli of patients with MCNS, the abundance of RelA was lower (Figure 2). These results suggest that induction of c-mip in podocytes is associated with a down-regulation of RelA.
      Figure thumbnail gr1
      Figure 1Induction of c-mip in MCNS relapse is associated with a down-regulation of RelA. Representative immunohistochemical analysis of serial sections from normal human kidney (NHK) and kidney biopsy specimens. A: Expression of c-mip. Note that c-mip was below detection limits in normal glomeruli, whereas it was clearly visualized along the external side of the capillary loops of MCNS relapse. The expression of c-mip was not restricted to cytoplasm compartment but it is also distributed in the nuclei. Note that c-mip was not detected in IgA nephropathy or in proliferative lupus nephritis (LN-IV). Scale bars: 20 μm. B: Expression of RelA in NHK and MCNS. Note that RelA is highly abundant in normal podocytes, whereas it was reduced in MCNS relapse. Scale bars: 20 μm.
      Figure thumbnail gr2
      Figure 2Confocal microscopy analysis. Detection of RelA (green); nephrin, tie-2, vimentin, and lamin-B (red) in normal human kidney (NHK, left) and in kidney biopsy specimens from MCNS relapse (right). Note that RelA partially colocalized with nephrin and lamin, but not with vimentin and tie-2 in normal glomeruli. The expression of RelA was hardly detected in MCNS relapse. Scale bars: 20 μm.

       Expression of c-mip in Vivo Induces a Down-Regulation of RelA at the Posttranscriptional Level

      To understand the functional consequences of c-mip induction in podocytes, we generated transgenic mice expressing c-mip under nephrin promoter to restrict transgene expression to podocytes. c-mip transgenic mice develop heavy proteinuria with foot process effacement without inflammatory lesions or immune complex deposits.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      The abundance of the RelA transcripts measured by quantitative RT-PCR did not significantly differ between wild-type and transgenic mice (Figure 3A). By contrast, Western blot analysis of glomerular protein lysates showed that RelA abundance was significantly decreased in c-mip transgenic mice, as compared with wild-type mice (Figure 3B), which suggests that c-mip affects only the stability of the RelA protein. Indeed, co-culture of c-mip–transfected podocytes in the presence of lactacystin, a potent proteasome inhibiter, prevented the decrease of endogenous RelA (Figure 3C), suggesting that c-mip targets RelA through proteasome-mediated degradation.
      Figure thumbnail gr3
      Figure 3The RelA protein is destabilized in the presence of c-mip, in vivo and in vitro. Glomeruli from wild-type (WT, n = 5) and 6-month-old c-mip transgenic mice [Tg(+), n = 5] were isolated, and the abundance of RelA was analyzed. A: Quantification of RelA transcripts by RT-qPCR. B: Quantitative Western blot analysis of RelA protein from WT and Tg(+) mice. Data are representative of three independent experiments. C: Podocytes were transiently transfected with c-mip or empty vector (Ev), cultured 20 hours with or without lactacystin (2 mmol/L), then analyzed by Western blotting. Note that lactacystin prevents the destabilization of endogenous RelA. Data are representative of two independent experiments.

       c-mip Displays Proapoptotic Properties

      On the basis of the studies performed in NF-κB–deficient mice, it is believed that NF-κB is a potent antiapoptotic transcription factor.
      • Gerondakis S.
      • Grumont R.
      • Gugasyan R.
      • Wong L.
      • Isomura I.
      • Ho W.
      • Banerjee A.
      Unravelling the complexities of the NF-kappa B signalling pathway using mouse knockout and transgenic models.
      Given our findings showing a down-regulation of RelA, which is essential for NF-κB activity, we sought to determine whether c-mip influences the signaling pathways involved in apoptosis. Apoptosis is defined by morphological features including loss of adhesion, cell shrinkage, and biochemical alterations such as up-regulation of Bax and caspase-3 activity. Bax belongs to the Bcl-2 family of proteins, which include both antiapoptotic (eg, Bcl-2 and Bcl-xl) and proapoptotic (eg, Bax, Bad) members.
      • Youle R.J.
      • Strasser A.
      The BCL-2 protein family: opposing activities that mediate cell death.
      Caspase-3 is one of the final downstream effectors of the apoptotic process, and its activation is closely associated with mitochondrial dysfunction.
      • Wada T.
      • Pippin J.W.
      • Marshall C.B.
      • Griffin S.V.
      • Shankland S.J.
      Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins.
      To assess whether c-mip is a proapoptotic molecule, we first transiently transfected c-mip alone in podocyte cell line and analyzed the expression level of Bax, BclII, and caspase-3. Overexpression of c-mip induced an up-regulation of Bax and caspase-3, whereas BclII abundance decreased when compared with empty vector–transfected cells (Figure 4A). On the other hand, cotransfection of RelA antagonized the proapoptotic effects of c-mip. We then sought to find whether these changes are reproducible in differentiated podocytes stably transfected with c-mip or empty vector. We studied five independent c-mip stably transfected podocyte cell lines and two control clones stably transfected with empty vector. We performed quantitative Western blot analysis to measure the abundance of the Bcl-2 and Bax proteins that were normalized to GAPDH loading. As shown in Figure 4B, Bax was overproduced in c-mip–overexpressing podocytes, so that the Bax/Bcl-2 ratio was significantly increased (Figure 4B). Morphological analyses showed that empty vector–transfected cells displayed small morphological changes, whereas c-mip–overexpressing podocytes exhibited severe cell shrinkage and most cells had detached. To assess whether these morphological changes involve caspase-3 activation, we performed a caspase-3 activity assay on stable transfectant cells. Podocytes overexpressing c-mip exhibited an activation of caspase-3 in a time-dependent manner, whereas no significant change was detected in the empty vector–transfected podocytes (Figure 4C).
      Figure thumbnail gr4
      Figure 4Transient and stable overexpression of c-mip in podocytes promotes apoptosis. A: Podocytes were singly transiently transfected with empty vector (Ev) or c-mip or cotransfected with Ev/c-mip or RelA/c-mip. The expression of Bax, BclII, and caspase-3 was analyzed by Western blotting on the protein lysates. Note that c-mip increases Bax and caspase-3 and down-regulates BclII, these alterations being prevented by the coexpression of RelA. B: Podocytes were stably transfected with c-mip expression plasmid (pDest 40) or empty vector (Ev), then cloned by limiting dilution. Morphological and/or quantitative Western blot data for six clones (five c-mip and one Ev clones) are shown. c-mip was revealed by anti-V5 tag monoclonal antibody. Quantification of c-mip was performed by subtracting the background related to the empty vector. Note that c-mip abundance varies depending on the clone; higher c-mip abundance is correlated with severe cellular injury and an increase of the Bax/Bcl-2 ratio. C: Differentiated podocytes stably transfected with c-mip or empty vector (Ev) were tested for caspase-3 activity assays. The caspase-3 activity was monitored every 30 minutes for 7 hours.
      The relevance of these findings was analyzed in c-mip transgenic mice. In this model, mice develop after 3 months of age progressive FSGS.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      Histological analysis (Figure 5A) showed that the number of glomeruli displaying FSGS lesions was increased from 3 (4.0% ± 1.5%), 6 (7.5% ± 2.5%) to 12 months of age (23.0% ± 3.5%). Western blot analysis from glomerular extracts revealed increased Bax in c-mip Tg mice, whereas Bcl-2 was reduced, with a Bax/Bcl-2 ratio that was increased by 3.1 fold, as compared with wild-type mice (Figure 5B). We then used the TUNEL assay to detect DNA fragmentation in podocytes in vivo. In wild-type mice and in 3-month-old transgenic mice, TUNEL-positive nuclei were not detectable in glomeruli. However, in transgenic mice of 6 and 12 months of age, a number of podocytes became positive for TUNEL (Figure 6). Collectively, these results suggest that c-mip promotes apoptosis in vitro and in vivo.
      Figure thumbnail gr5
      Figure 5c-mip promotes progressive FSGS in transgenic mice. A: Representative Periodic acid-Schiff (PAS) staining of kidney sections from 3- to 12-month-old wild-type and proteinuric Tg mice. Scale bars: 50 μm (top of each set); 20 μm (bottom of each set). Glomerular alterations are apparent at 6 and 12 months, characterized by FSGS lesions and mesangial expansion. The tubules appear normal, and no inflammatory injuries can be seen. Right, percentage of glomeruli with FSGS. Five mice were analyzed by age range (≥300 glomeruli). Data represent the mean ± SEM. B: Glomerular protein extracts from 12-month-old wild-type and proteinuric Tg mice were analyzed by Western blotting for the relative expression of Bax and Bcl-2. Quantitative data for Bax/Bcl-2 ratio are shown to the right.
      Figure thumbnail gr6
      Figure 6c-mip promotes apoptosis in vivo. TUNEL assay on paraffin kidney sections of wild-type and c-mip transgenic mice at 3, 6, and 12 months of age. Note that TUNEL-positive cells were not detected in 3-month-old transgenic mice, whereas many podocytes became positive at 6 months of age. Scale bars: 20 μm.
      FSGS lesions are characterized by dysregulation of podocyte function, leading to apoptosis. We have previously shown that c-mip abundance is increased in primary FSGS. Therefore, we investigated whether FSGS biopsy specimens exhibit active, cleaved caspase-3, a marker of cell apoptosis. Immunochemistry analysis was performed in seven FSGS biopsy specimens. Active caspase-3 was detected in all examined cases, to a variable degree, along the external side of some capillary loops, consistent with podocyte localization (Figure 7). The expression was decreased or disappeared in podocyte-depleted areas corresponding to severe FSGS lesions or glomerulosclerosis (Figure 7). Interestingly, c-mip was increased in glomeruli in a pattern similar to that observed with active caspase-3.
      Figure thumbnail gr7
      Figure 7Expression of c-mip and caspase-3 in FSGS biopsies. Representative immunostaining of c-mip and cleaved caspase-3 from FSGS biopsy specimens.
      • Mathieson P.W.
      Minimal change nephropathy and focal segmental glomerulosclerosis.
      • Tryggvason K.
      • Patrakka J.
      • Wartiovaara J.
      Hereditary proteinuria syndromes and mechanisms of proteinuria.
      • Shankland S.J.
      The podocyte's response to injury: role in proteinuria and glomerulosclerosis.
      • Shih V.F.
      • Tsui R.
      • Caldwell A.
      • Hoffmann A.
      A single NF-kappa B system for both canonical and non-canonical signaling.
      In most cases, the localization of c-mip and active caspase-3 was observed in close proximity. The staining strongly decreases or disappears in podocyte-depleted areas corresponding to FSGS lesions or severe glomerulosclerosis. Scale bars: 20 μm.

      Discussion

      The role of NF-κB in glomerular biology and in pathophysiology of podocyte disease remains little explored. In the present work, we provide evidence that i) increased expression of c-mip in podocytes of patients with INS is correlated with a down-regulation of RelA; ii) c-mip destabilizes endogenous RelA protein in vitro, which is prevented by lactacystin, suggesting that c-mip targets RelA through proteasome-mediated degradation; iii) stable transfection of c-mip in the podocytes is associated with cell shrinkage and increased abundance of Bax and active caspase-3; iv) c-mip induces in vivo an up-regulation of Bax and a decrease of Bcl-2, supporting the role of c-mip in apoptosis; v) c-mip transgenic mice develop progressive FSGS in the absence of overt tubular and interstitial lesions; and vi) the abundance of c-mip and active caspase-3 was increased in human primary FSGS.
      We have previously shown that c-mip interacts through its pleckstrin homology domain with Fyn and PI3 kinase regulatory subunit (p85), and exerts an inhibitory role in proximal signaling.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      • Kamal M.
      • Pawlak A.
      • BenMohamed F.
      • Valanciute A.
      • Dahan K.
      • Candelier M.
      • Lang P.
      • Guellaen G.
      • Sahali D.
      C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase.
      c-mip also binds to and inhibits the activation of neuronal Wiskott–Aldrich syndrome protein (N-WASP), a major component of cytoskeleton that regulates the ARP2/3-mediated actin nucleation. In addition, we found that c-mip induces in vitro and in vivo cytoskeleton disorganization,
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      and increases the abundance of death-associated protein (DAP) kinase.
      • Kamal M.
      • Pawlak A.
      • BenMohamed F.
      • Valanciute A.
      • Dahan K.
      • Candelier M.
      • Lang P.
      • Guellaen G.
      • Sahali D.
      C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase.
      DAP kinase promotes apoptosis by disrupting integrin-mediated cell adhesion, alteration of cytoskeleton, and loss of extracellular matrix–dependent survival signals.
      • Wang W.J.
      • Kuo J.C.
      • Yao C.C.
      • Chen R.H.
      DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals.
      Alteration of the actin network induces the production of reactive oxygen species from mitochondria, leading to apoptosis.
      • Gourlay C.W.
      • Carpp L.N.
      • Timpson P.
      • Winder S.J.
      • Ayscough K.R.
      A role for the actin cytoskeleton in cell death and aging in yeast.
      We have previously found that c-mip binds RelA through its leucine-rich repeat–containing C-terminal domain.
      • Kamal M.
      • Valanciute A.
      • Dahan K.
      • Ory V.
      • Pawlak A.
      • Lang P.
      • Guellaen G.
      • Sahali D.
      C-mip interacts physically with RelA and inhibits nuclear factor kappa B activity.
      We show here that c-mip may target RelA to proteasome-dependent degradation. Altogether, our results suggest that c-mip is a multifunctional protein and may promote apoptosis through different mechanisms, including cytoskeleton alterations, up-regulation of DAP kinase, and down-regulation of RelA.
      Several studies have shown that NF-κB hyperactivation is mainly correlated with the intensity of proteinuria and is restricted in the tubules of patients with progressive kidney diseases, including glomerular diseases.
      • Mezzano S.A.
      • Barria M.
      • Droguett M.A.
      • Burgos M.E.
      • Ardiles L.G.
      • Flores C.
      • Egido J.
      Tubular NF-kappa B and AP-1 activation in human proteinuric renal disease.
      • Ashizawa M.
      • Miyazaki M.
      • Abe K.
      • Furusu A.
      • Isomoto H.
      • Harada T.
      • Ozono Y.
      • Sakai H.
      • Koji T.
      • Kohno S.
      Detection of nuclear factor-kappa B in IgA nephropathy using Southwestern histochemistry.
      • Zheng L.
      • Sinniah R.
      • Hsu S.I.
      In situ glomerular expression of activated NF-kappa B in human lupus nephritis and other non-proliferative proteinuric glomerulopathy.
      Indeed, NF-κB activity is associated with increased tubular expression of the chemokines monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), osteopontin, and the profibrogenic cytokines platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGFβ),
      • Mezzano S.A.
      • Barria M.
      • Droguett M.A.
      • Burgos M.E.
      • Ardiles L.G.
      • Flores C.
      • Egido J.
      Tubular NF-kappa B and AP-1 activation in human proteinuric renal disease.
      leading to tubular apoptosis and fibrosis. By contrast, these studies fail to detect significant hyperactivation of NF-κB in podocytes in certain forms of glomerular diseases, suggesting that this mechanism does not prevail in these cells. It has been reported that NF-κB is expressed in a few mesangial cells and tubular epithelial cells in MCNS and membranous nephropathy, whereas it is detected in podocytes of IgA nephropathy and non-IgA mesangial proliferative glomerulonephritis.
      • Ashizawa M.
      • Miyazaki M.
      • Abe K.
      • Furusu A.
      • Isomoto H.
      • Harada T.
      • Ozono Y.
      • Sakai H.
      • Koji T.
      • Kohno S.
      Detection of nuclear factor-kappa B in IgA nephropathy using Southwestern histochemistry.
      These data are consistent with our results because the up-regulation of c-mip might account for the nondetection of NF-κB in podocytes in INS. Conversely, in IgA nephropathy, where the expression of NF-κB is found increased, c-mip was not detected in podocytes, in accord with previous data.
      • Zhang S.Y.
      • Kamal M.
      • Dahan K.
      • Pawlak A.
      • Ory V.
      • Desvaux D.
      • Audard V.
      • Candelier M.
      • Mohamed F.B.
      • Matignon M.
      • Christov C.
      • Decrouy X.
      • Bernard V.
      • Mangiapan G.
      • Lang P.
      • Guellaen G.
      • Ronco P.
      • Sahali D.
      c-mip impairs podocyte proximal signaling and induces heavy proteinuria.
      The down-regulation of RelA in INS relapse may explain why these podocyte diseases are not associated with inflammatory processes in glomeruli. By contrast, activation of RelA in the podocytes of Par4-deficient mice is associated with severe glomerular and tubular inflammatory lesions.
      • Hussain S.
      • Romio L.
      • Saleem M.
      • Mathieson P.
      • Serrano M.
      • Moscat J.
      • Diaz-Meco M.
      • Scambler P.
      • Koziell A.
      Nephrin deficiency activates NF-kappa B and promotes glomerular injury.
      Moreover, in pathological situations associated with sustained c-mip increase, the down-regulation of NF-κB in glomeruli might represent a possible mechanism involved in podocyte apoptosis and may contribute to podocyte depletion in some podocyte diseases without early involvement of tubular and interstitial tissue injuries.
      Two distinct signaling pathways are involved in the initiation of apoptosis: the extrinsic apoptotic pathway or receptor-linked pathway requires the binding of a ligand to a death receptor on the cell surface, such as TNF receptor; and the intrinsic apoptotic pathway that is mediated by mitochondrial damage and release of cytochrome c.
      • Reed J.C.
      Mechanisms of apoptosis.
      The development of the signaling cascade leading to apoptosis requires in most situations the up-regulation of p53. Despite several assays, we did not observe an up-regulation of p53 in stable transfected podocytes overexpressing c-mip or in c-mip Tg mice (data not shown), which suggests that the proapoptotic function of c-mip does not require p53. Some studies have shown that the ability to trigger apoptotic pathways in the absence of p53 may result from the activation of extracellular signal-regulated kinase (Erk) signaling.
      • Tang D.
      • Wu D.
      • Hirao A.
      • Lahti J.M.
      • Liu L.
      • Mazza B.
      • Kidd V.J.
      • Mak T.W.
      • Ingram A.J.
      ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53.
      However, the Erk pathway has multiple functions, including pro- and antiapoptotic effects, of which the mechanisms are still unclear.
      • Wada T.
      • Penninger J.M.
      Mitogen-activated protein kinases in apoptosis regulation.
      Induction of apoptosis following exposition to puromycin aminonucleoside, in cultured podocytes as well as in vivo, in a model of FSGS in rats, is associated with an increase of Bax and a reduction of Bcl-2.
      • Wada T.
      • Pippin J.W.
      • Marshall C.B.
      • Griffin S.V.
      • Shankland S.J.
      Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins.
      • Wang W.
      • Tzanidis A.
      • Divjak M.
      • Thomson N.M.
      • Stein-Oakley A.N.
      Altered signaling and regulatory mechanisms of apoptosis in focal and segmental glomerulosclerosis.
      The underlying mechanism involves an up-regulation of p53 since, in this model, apoptosis is prevented by pifithrin-α (PFT-α) and dexamethasone, which inhibit p53 activation. Indeed, two signaling pathways have been involved in podocyte apoptosis induced by puromycin aminonucleoside, depending on caspase-3 or apoptosis-inducing factor (AIF), respectively. Dexamethasone inactivates AIF by inhibiting its nuclear translocation.
      • Wada T.
      • Pippin J.W.
      • Marshall C.B.
      • Griffin S.V.
      • Shankland S.J.
      Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins.
      Insulin growth factor-1 (IGF-1) protects from apoptosis induced by etoposide, an inhibitor of topoisomerase II, through a mechanism involving activation of PI3 kinase and Bad.
      • Bridgewater D.J.
      • Ho J.
      • Sauro V.
      • Matsell D.G.
      Insulin-like growth factors inhibit podocyte apoptosis through the PI3 kinase pathway.
      This conclusion is based on the fact that the inhibitors of PI3 kinase, Whormaninn and Ly294002, abrogate the antiapoptotic effect of IGF1. Moreover, nephrin and vascular endothelial growth factor (VEGF) inhibit apoptosis by recruiting the PI3 kinase-Akt signaling pathway.
      • Foster R.R.
      • Saleem M.A.
      • Mathieson P.W.
      • Bates D.O.
      • Harper S.J.
      Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes.
      Both the VEGF and nephrin signaling pathways appear interconnected because mutation of nephrin, which disrupts the nephrin signaling, abrogates the antiapoptotic effect of VEGF.
      • Foster R.R.
      • Saleem M.A.
      • Mathieson P.W.
      • Bates D.O.
      • Harper S.J.
      Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes.
      The antiapoptotic role of CD2-associated protein (CD2AP), an adapter protein anchoring the nephrin-mediated proximal signals to actin cytoskeleton, has been evidenced by the phenotype of CD2AP-deficient mice, which develop nephrotic syndrome and renal failure caused by glomerulosclerosis.
      • Shih N.Y.
      • Li J.
      • Karpitskii V.
      • Nguyen A.
      • Dustin M.L.
      • Kanagawa O.
      • Miner J.H.
      • Shaw A.S.
      Congenital nephrotic syndrome in mice lacking CD2-associated protein.
      In this model, proteinuria occurs in the context of accelerated apoptosis resulting from hyperactivation of p38 mitogen–activated protein kinase (p38 MAPK) by TGFβ1.
      • Schiffer M.
      • Bitzer M.
      • Roberts I.S.
      • Kopp J.B.
      • ten Dijke P.
      • Mundel P.
      • Bottinger E.P.
      Apoptosis in podocytes induced by TGF-beta and Smad7.
      • Schiffer M.
      • Mundel P.
      • Shaw A.S.
      • Bottinger E.P.
      A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis.
      The mechanism by which CD2AP impedes the apoptotic process involves a down-regulation of p38 MAPK via activation of PI3 kinase and Akt signaling.
      • Schiffer M.
      • Mundel P.
      • Shaw A.S.
      • Bottinger E.P.
      A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis.
      Direct evidence of the role of TGFβ1 in podocyte apoptosis comes from the study of transgenic mice overexpressing TGFβ1 under control of the albumin promoter.
      • Kopp J.B.
      • Factor V.M.
      • Mozes M.
      • Nagy P.
      • Sanderson N.
      • Bottinger E.P.
      • Klotman P.E.
      • Thorgeirsson S.S.
      Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease.
      In this model, Tg mice display glomerulosclerosis and interstitial fibrosis with renal failure, and death in 50% of offspring at 5 to 12 weeks of age.
      • Schiffer M.
      • Bitzer M.
      • Roberts I.S.
      • Kopp J.B.
      • ten Dijke P.
      • Mundel P.
      • Bottinger E.P.
      Apoptosis in podocytes induced by TGF-beta and Smad7.
      Histological lesions observed in TGFβ1 and c-mip Tg models are different. Glomerulosclerosis and extracellular matrix expansion are present in the early stages (∼2 weeks) in TGFβ1 Tg mice, whereas glomeruli are morphologically normal despite the presence of a nephrotic syndrome and foot process fusions in c-mip Tg mice, until 4 to 5 months of age. Renal failure and death are rare during the first year of life in c-mip Tg mice even though these complications are common in TGFβ1 Tg mice. However, as with c-mip Tg mice, podocyte apoptosis precedes mesangial expansion.
      In conclusion, we report here new functional aspects of c-mip in podocytes. We show that c-mip induces in vitro and in vivo the destabilization of the RelA protein in podocytes. The up-regulation of c-mip and active caspase-3 in human FSGS suggests that sustained up-regulation of c-mip might result in podocyte depletion and progressive FSGS.

      Acknowledgments

      We are grateful to Dr. Peter Mundel for providing us with the mouse podocyte cell line.

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