Apoptosis in podocytes induced by TGF-β and Smad7 (original) (raw)
TGF-β1 TG mice: a murine model to investigate mechanisms of progression of glomerulosclerosis in the kidney. Albumin/TGF-β1 TG mice are characterized by progressive renal disease induced by elevated circulating TGF-β1 (9). Circulating TGF-β1 levels are elevated at 2–3 weeks of age in TG mice and lead to progressive glomerulosclerosis and interstitial fibrosis with renal failure and death in approximately half of TG animals at 5–12 weeks of age.
To define phenotypic characteristics at early and advanced stages of glomerulosclerosis in this model, we examined six 2-week-old and six 5-week-old TG mice derived from multiple litters. As controls, we examined four 2-week- and four 5-week-old WT control mice. Glomerular pathology resembling glomerulosclerosis was detected in less than half of the examined glomeruli of 2-week-old TG mice (Table 1). At 5 weeks of age, TG mice had developed significant azotemia and albuminuria associated with global glomerulosclerosis with or without segmental accentuation in all glomeruli (Table 1). Glomeruli were normal in all WT mice. These results confirm that glomerular lesions typically identified at 2 weeks of age are representative of an early stage, and those typically identified at 5 weeks of age are representative of advanced stages, in the course of progressive glomerulosclerosis caused by TGF-β1 in TG mice.
Blood urea nitrogen (BUN), albuminuria, and glomerulosclerosis in TG and WT control mice
Expression of Smad7 is upregulated in podocytes and decreased in the mesangium in TGF-β1 TG mice. We observed previously that whole kidney mRNA levels of inhibitory Smad7 were elevated in TG mice with moderate glomerulosclerosis and interstitial fibrosis (M. Schiffer and E. Böttinger, unpublished observations). To determine the glomerular cell type(s) associated with increased Smad7 expression, we performed immunofluorescence double labeling on renal cortex sections. Smad7 protein was expressed predominantly in endocapillary/mesangial areas in WT kidney, whereas only few of the podocytes per section were labeled by Smad7 antibody (Figure 1, a–c). In contrast, most podocyte cell bodies were strongly labeled by Smad7 antibody, whereas only few endocapillary/mesangial cells expressed Smad7 protein in 2-week-old TG mice (Figure 1, d–f). Our data suggest that Smad7 protein expression is induced in podocytes in 2-week-old TG mice, whereas endocapillary/mesangial expression of Smad7, normally observed in WT mice, is decreased in TG mice at this age.
(a–f) Indirect immunofluorescence of mouse renal cortex sections. Mouse anti-synaptopodin staining (a and d) visualized with FITC-conjugated anti-mouse IgG; rabbit anti-Smad7 (b and e) visualized with Cy3-conjugated anti-rabbit IgG in 2-week-old WT (a–c) and TG mice (d–f). Podocytes, blue arrows; endocapillary/mesangial cells, white arrows; artifact (red blood cell), yellow arrows. (g–i) Mouse anti-smooth muscle actin (α-SMA) IgG labeling (g) and anti-Smad7 staining (h). (j) Northern blot shows Smad7 mRNA levels after TGF-β treatment in podocytes cultured under permissive (33°C) or nonpermissive (37°C) conditions. GAPDH is shown for loading control.
To examine whether TGF-β1 activates Smad7 synthesis in podocytes, we used a conditionally immortalized murine podocyte cell line (10). TGF-β1 rapidly induced Smad7 mRNA levels in podocytes, as has been shown previously in other cell types (15) (Figure 1j). Immunoblots for Smad7 protein showed similar results (data not shown).
Smad7 expression coincides with podocyte damage at an early stage of glomerulosclerosis and is associated with progressive podocyte depletion in TGF-β1 TG mice. To establish semiquantitative measurements of podocyte damage and podocyte numbers and to examine associations with Smad7 expression, we devised a staining strategy combining PAS staining with immunoperoxidase staining for Smad7. Criteria for podocyte damage included pseudocyst formation and partial or complete detachment from basement membrane. Representative glomerular sections in each category are shown in Figure 2. Results are summarized in Table 2.
(a–f) PAS staining and anti-Smad7 immunoperoxidase labeling of renal cortex sections of WT (a and d) and TG mice (b and e) at 2 weeks (TG 2 wk) (a–c) and 5 weeks (TG 5 wk) (d–f) of age. Absence of Smad7 immunoperoxidase labeling in the presence of blocking peptide in 2-week-old (c) and 5-week-old (f) TG mice as control for specificity of staining. Dotted arrows depict Smad7-negative podocytes and stars denote Smad7-positive mesangial areas (a and d). Arrows indicate Smad7-positive podocytes with representative features of damage, including pseudocyst formation and partial or complete detachment from basement membrane (b and e). Arrowhead shows synechiae of basement membrane and Bowman’s capsule (e). (g) Histogram shows average numbers of podocytes (filled bars) and Smad7-positive podocytes (hatched bars) per central glomerular section. Bars represent average ± SEM numbers of cells per glomerular section determined from 180 glomeruli (30 per mouse) in TGF-β1 TG mice at 2 weeks and at 5 weeks of age, respectively, and 240 glomeruli (30 per mouse) in the WT. Results of t test: *Podocyte counts in WT vs. TG 2 wk, P < 0.001; +Smad7-positive podocyte counts in WT vs. TG 2 wk; **podocyte counts in WT vs. TG 5 wk, P < 0.001; ++Smad7-positive podocyte counts in WT vs. TG 5 wk, P < 0.001. (h) Histogram shows numbers of podocytes with criteria of injury (filled bars) and Smad7-positive injured podocytes (hatched bars). Annotations are as described in g. (i) Histogram shows glomerular surface area (filled bars) and mesangial surface area (hatched bars) per glomerular section in arbitrary units (annotations as in g). Line graph indicates ratio of Smad7-positive mesangial area to total mesangial area (see Methods for definitions).
Summary of analyses of cell counts and surface areas in glomerular sections
Average counts of podocytes per glomerular section were not significantly different between 2-week-old and 5-week-old WT mice, but were significantly reduced in 2-week-old TG compared with WT mice, and were further reduced in 5-week-old TG mice (Table 2). Counts of Smad7-positive podocytes were significantly increased in 2-week-old TG compared with WT mice. In 5-week-old TG mice, all podocytes were expressing Smad7. Average counts of damaged podocytes were 14-fold increased in 2-week-old TG compared with WT mice, but not significantly different from age-matched WT mice in 5-week-old TG mice (Table 2). Nearly all damaged podocytes expressed Smad7 in 2-week-old and 5-week-old TG mice. Our results demonstrate that morphological signs of cellular damage are detectable in podocytes at early stages of progressive glomerulosclerosis and are associated with Smad7 protein synthesis in these cells.
Mesangial expansion manifests at an advanced stage of glomerulosclerosis and is associated with loss of Smad7 expression. Given that podocyte damage was apparent at early stages in our model, we wanted to determine whether it was coincident with, or preceded, mesangial expansion. We used NIH Image analysis software (version 6.1; NIH, Bethesda, Maryland, USA) to compute total glomerular section surface area and mesangial section surface area by outlining entire glomerular tuft, or mesangial segments minus capillary lumen of glomerular tufts. Average glomerular area in arbitrary units was not significantly different between 2-week-old and 5-week-old WT mice, but was increased in 2-week-old TG compared with WT mice, and was further increased in 5-week-old TG mice (Table 2). Average mesangial area per glomerular section was not significantly different between 2-week-old and 5-week-old WT mice or 2-week-old TG mice, but was considerably increased in 5-week-old TG mice (Table 2). These data indicate that mesangial expansion manifests itself during advanced stages of glomerulosclerosis in TG mice. To estimate the extent of Smad7 expression in mesangial areas in TG and WT mice, we determined immunoperoxidase-stained mesangial areas as a fraction of total mesangial area on each examined glomerular section. By this measure, the fractions of Smad7-positive mesangial areas were significantly reduced in both, 2-week-old and 5-week-old TG mice when compared with WT mice (Table 2).
Apoptosis is increased at early stages of progressive glomerulosclerosis in podocytes and at advanced stages in other glomerular cells in TGF-β1 TG mice. To begin to explore the underlying mechanisms of podocyte damage and depletion in our model, we determined rates of apoptosis in podocytes and endocapillary/mesangial cells in WT and TG mice. Podocytes were scored as apoptotic if nuclear labeling by DAPI (blue), cytoplasmic synaptopodin labeling (red), and nuclear TUNEL labeling (green) resulted in turquoise nuclei with red rim (Figure 3, a–l, blue arrows). Overlap of DAPI labeling and TUNEL labeling in the absence of synaptopodin labeling was scored as apoptotic glomerular cells other than podocytes (Figure 3, a–l, white arrows). Colocalizations of synaptopodin labeling and TUNEL staining in the absence of DAPI labeling resulted in yellow signals and were caused by artifactual staining of red blood cells (Figure 3, a–l, yellow arrows). There was a 20-fold increase in the average of TUNEL positive podocytes per glomerular section in 2-week-old TG mice compared with WT, and a fourfold increase in 5-week-old TG mice (Table 2; Figure 3m). Rates of apoptosis in endocapillary/mesangial cells were not significantly different between WT and 2-week-old TG mice, but were sixfold increased in 5-week-old TG mice. These results demonstrate that increased rates of apoptosis in podoctyes were characteristic for early stages of glomerulosclerosis in this model, and coincided with increased rates of damage in podocytes as determined by morphological criteria (see Figure 2). In contrast, rates of apoptosis in endocapillary/mesangial cells were significantly increased at advanced stages of glomerulosclerosis.
(a–l) Triple fluorescence labeling using DAPI (a, e, and i), rabbit anti-synaptopodin IgG (b, f, and j), and TUNEL assay (c, g, and k) in renal cortex sections of WT (a–d), 2-week-old TG (e–h), and 5-week-old TG (i–l) mice. TUNEL-positive and DAPI-negative red blood cells (artifacts), yellow arrows; TUNEL-positive podocytes, blue arrows; TUNEL-positive endocapillary/mesangial cells, white arrows. Representative results are shown. (m) Average ± SEM of TUNEL-positive podocytes (black bars) and TUNEL-positive endocapillary/mesangial cells (gray bars) per glomerular section. Data for 2-week-old and 5-week old TG are combined. *TUNEL-positive podocytes in WT vs. 2-week-old TG, P < 0.001. **Podocytes in WT vs. 5-week-old TG, P < 0.05. +Endocapillary/mesangial cells in WT vs. 5-week-old TG, P < 0.01. NS, not significant.
TGF-β1–induced apoptosis in podocytes is associated with increased Bax protein synthesis and caspase-3 activity. We used a conditionally immortalized murine podocyte cell line (10) to explore molecular mechanisms of podocyte apoptosis. Podocytes maintained in both permissive and nonpermissive culture conditions were incubated with TGF-β1 for 2 days before DAPI and TUNEL staining. Podocytes with characteristic morphological features of apoptosis, including condensed nuclei and/or fragmented nuclei (16), and TUNEL-positive nuclei were significantly more common in TGF-β–treated compared with untreated podocytes, irrespective of culture conditions (Figure 4a). TGF-β induced characteristic DNA-fragmentation as early as 24 hours after treatment (Figure 4b). Addition of an inhibitor of caspase-3, Z-Val-Ala-Asp(Ome)-FMK (zVAD-fmk), prevented DNA-fragmentation induced by TGF-β1 (Figure 4b), indicating that TGF-β1 caused apoptosis through activation of effector caspases.
(a) Bars indicate number of podocytes (mean ± SD) with apoptotic nuclei per 100 total cells as quantitated by TUNEL assay. Analysis was performed at permissive (33°C) and nonpermissive (37°C) conditions in the absence or presence of TGF-β1, respectively. (b) Ethidium bromide gel electrophoresis shows DNA fragmentation (laddering) in podocytes cultured under permissive conditions (33°C) or nonpermissive conditions (37°C). Cells were maintained without (–) or with (+) TGF-β1 (1 ng/ml) as indicated, in the absence (–) or presence (+) of caspase inhibitor zVAD-fmk. (c) Immunoblotting detecting Bax, pro–caspase-3, 113-kDa PARP, and 85-kDa PARP cleavage product. GDP dissociation inhibitor (GDI) is shown for loading control.
To examine further potential mediators of TGF-β1–induced apoptosis, we analyzed the abundance of Bcl-2 family proteins (Bcl-2, Bcl-X[L], Bad and Bax) and of procaspase-3 by Western blot analysis. Proapoptotic Bcl2-associated X protein (Bax) expression was increased at 2– 6 hours, followed by a decrease in procaspase-3 levels at 6–48 hours of TGF-β treatment. Induction of caspase-3 activity by TGF-β was confirmed using a fluorimetric assay (Figure 5c). Levels of intact 113-kDa PARP, a substrate of activated caspases, were decreased at 24 and 48 hours, coincident with the appearance of 85-kDa PARP cleavage products (Figure 4c). Together, these data suggest that TGF-β induces apoptosis in podocytes through increased synthesis of the proapoptotic protein Bax and activation of effector caspase-3.
(a) Western blot demonstrates levels of Flag-Smad7 in podocytes maintained under permissive conditions. Adenoviral vectors containing either control LacZ or Flag-Smad7 cDNAs were used to infect cells at various moi’s, as indicated. (b) Histogram shows the normalized average numbers of apoptotic cells visualized by DAPI per hpf (in 50 hpf total) from a representative experiment. Podocyte cultures were infected with AdLacZ or AdSmad7 adenoviral vectors and left untreated or treated with TGF-β1 in the absence or presence of caspase-3 inhibitor zVAD-fmk. Results were normalized for cell density. (c) Relative enzymatic activity of caspase-3 measured as fluorochrome release at 460 nm in infected podocytes cultured under permissive conditions in the absence (–) or presence (+) of TGF-β1. Enzyme activity is normalized to uninfected podocytes (set at 1). (d) Histogram shows the normalized average numbers of apoptotic cells as detected by TUNEL assay per hpf (in 50 hpf total) from a representative experiment. Podocyte cultures were infected with AdLacZ or AdSmad7 adenoviral vectors and left untreated or treated with TGF-β in the presence of control mouse immunoglobulin (IgG) or panneutralizing anti–TGF-β1 antibody (2G7). Results were normalized for total cell density.
Increased expression of Smad7 is sufficient to induce apoptosis in podocytes through caspase-3–independent pathways. Because we showed that Smad7 expression was increased in injured podocytes in situ in TGF-β TG mice, and that TGF-β stimulated Smad7 synthesis in podocyte cultures, we examined whether Smad7 is able to increase apoptosis in podocytes independently of TGF-β. We used an adenoviral expression system for flag-epitope–tagged Smad7 (AdSmad7) (13) to achieve efficient expression of Flag-Smad7 in infected podocytes, as verified by immunoblotting (Figure 5a) and by indirect immunofluorescence demonstrating infection efficiencies of more than 98% without detectable cellular toxicity at moi’s of 100–200 (data not shown). Cellular toxicity and apoptosis were observed at higher moi. Enhanced expression of Smad7 resulted in significantly increased rates of apoptotic nuclei in AdSmad7-infected podocytes, when compared with AdLacZ control infection (Figure 5b). Caspase-3 inhibitor zVAD-fmk had no effect on increased apoptotic rates induced by Smad7 expression, but significantly inhibited TGF-β–mediated increase in apoptotic rates. The proapoptotic effects of Smad7 expression and of TGF-β were additive (Figure 5b). To verify these results, we used a fluorimetric assay that measures caspase-3 activity (17). Although TGF-β treatment of podocytes significantly increased caspase-3 activity, Smad7 expression had no effect on baseline activity of caspase-3 (Figure 5c). In contrast with TGF-β (see Figure 4c), Smad7 expression had no effect on Bax and pro–caspase-3 protein levels (data not shown). To examine whether the proapoptotic activity of Smad7 required autocrine/paracrine activation of TGF-β, we repeated experiments for quantitation of apoptotic rates in podocyte cultures in the presence of a neutralizing mAb (2G7) against all three TGF-β isoforms (18). Presence of neutralizing anti–TGF-β antibody had no effect on Smad7-mediated apoptosis, but completely inhibited TGF-β–induced apoptosis in podocytes (Figure 5d). Together, these results demonstrate that Smad7 expression potentiates apoptosis in podocytes through caspase-3–independent and TGF-β–independent mechanisms, whereas TGF-β induces apoptosis in podocytes through activation of effector caspase-3. These results are also consistent with the additive proapoptotic effects of Smad7 and TGF-β1 in podocytes (Figure 5b).
MAP kinase p38 signaling is required for induction of apoptosis by TGF-β, but not by Smad7. Because we demonstrated that Smad7 expression inhibits Smad3/4-dependent transcriptional activation of reporter genes by TGF-β (M. Schiffer and E. Böttinger, unpublished data) whereas it augments TGF-β–induced apoptosis in podocytes, we examined whether Smad-independent signal transducers including proapoptotic p38 MAP kinase were required for the apoptotic response. Immunoblot analysis using monoclonal anti-phospho-p38 antibody confirmed activation of p38 MAP kinase by TGF-β after 20 minutes of treatment (Figure 6a). In contrast, we were unable to detect an effect of Smad7 expression on p38 phosphorylation in transduced podocytes (data not shown). Next, we repeated experiments to quantitate Smad7- and TGF-β–induced apoptosis in podocytes in the absence or presence of a chemical inhibitor of p38 (SB203580). SB203580 had no effect on apoptosis induced by Smad7 expression, although it blocked TGF-β–induced apoptosis completely (Figure 6b). These results suggest that MAP kinase p38 signaling is required for induction of apoptosis by TGF-β, but not by Smad7, in podocytes.
(a) Immunoblot demonstrates levels of phosphorylated p38 MAP kinase (pp38) in podocytes treated with LPS as positive control or TGF-β1 for various time intervals. (b) Histogram shows the normalized average numbers of apoptotic cells as detected by TUNEL assay per hpf (in 50 hpf total) from a representative experiment. Podocyte cultures were infected with AdLacZ or AdSmad7 adenoviral vectors and left untreated or treated with TGF-β in the absence or presence of p38 MAP kinase inhibitor SB203580. Results were normalized for total cell density. (c) Detection of the NF-κB p65-subunit (anti-p65) by indirect immunofluorescence in podocytes transiently cotransfected with green fluorescent protein expression plasmid pEGFP together with either empty control vector pcDNA3 or Smad7 expression vector pSmad7. Cells were either left untreated or treated with TNF-α for 30 minutes. Arrows indicate GFP and anti-p65 signals in pEGFP/pSmad7-cotransfected cells. (d) Bar graph showing normalized luciferase activity (RLU) mediated by the NF-κB–responsive reporter gene construct NF-κB-luc in podocytes cotransfected with pcDNA3 empty control or pSmad7 expression vectors. Cells were either left untreated or stimulated with TNF-α (10 ng/ml) after transfection. (e) Schematic demonstration of a new working model for proapoptotic signaling pathways induced by TGF-β and Smad7.
Smad7 expression in podocytes inhibits basal and inducible nuclear translocation and transcriptional activator function of anti-apoptotic survival factor NF-κB/p65. NF-κB/p65 is a signal transducer/transcriptional activator complex with well-established functional roles as anti-apoptotic cell survival factor (19). Recent reports indicate that inhibition of NF-κB/p65 causes spontaneous apoptosis independent of caspase activity in lymphoblastoid cells (20) and also suggest a potential role for Smad7 in inhibition of NF-κB (21). Thus, we reasoned that Smad7 may stimulate apoptosis in podocytes by blocking NF-κB/p65 activity. Podocytes were transiently cotransfected with green-fluorescent protein vector pEGFP together with either pcDNA3 control or pSmad7 expression vectors. Immunofluorescence analysis using anti-p65 antibody demonstrated that nuclear p65 labeling was significantly reduced in pSmad7-transfected podocytes at baseline and after stimulation with TNF-α, a major activator of cytoplasmic-to-nuclear translocation of NF-κB/p65 (Figure 6c). Next, we cotransfected a NF-κB/p65–responsive luciferase reporter gene construct (14) together with either pcDNA3 control vector or pSmad7 expression vector to examine whether Smad7 expression could modulate transcriptional activity of NF-κB/p65. Both basal and TNF-α–inducible NF-κB/p65 reporter gene activity was strongly inhibited in pSmad7 cotransfected podocytes compared to pcDNA3-transfected controls (Figure 6d). TGF-β had no significant effect on NF-κB/p65 reporter gene activity (data not shown). Our data suggest that expression of Smad7 inhibits cytoplasmic-to-nuclear translocation and transcriptional activator function of the anti-apoptotic NF-κB/p65 cell survival factor in podocytes.